Research ArticleCYTOKINES

Human IFN-γ immunity to mycobacteria is governed by both IL-12 and IL-23

See allHide authors and affiliations

Science Immunology  21 Dec 2018:
Vol. 3, Issue 30, eaau6759
DOI: 10.1126/sciimmunol.aau6759
  • Fig. 1 Identification of homozygous complete LOF IL-12Rβ2 Q138X and IL-23R C115Y mutations in families with MSMD.

    (A and B) Pedigrees of the two kindreds studied in this report. The gene and mutation are indicated under the kindred name. Solid black symbols indicate patients with MSMD, and solid gray symbols indicate cases of primary tuberculosis during childhood. Symbols linked with a double line indicate consanguinity. The genotype is indicated under each symbol, with M corresponding to the mutation found in each kindred. Arrows indicate the index case in each family. (C and D) Schematic representation of IL-12Rβ2 (C) and IL-23R (D). Rectangles represent individual exons of the gene, with the exon numbers indicated within the rectangle. In each case, the N-terminal portion of the protein is the extracellular domain, and gray-shaded areas represent the transmembrane domain. The mutations studied here are indicated below each protein in the corresponding exons. (E) HEK293T [human embryonic kidney (HEK) 293T] cells were either left untransfected (UT) or transfected with an empty vector (EV), a vector containing the C-terminal V5-tagged WT or Q138X mutant versions IL12RB2. Western blotting was performed with antibodies against the V5 tag or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. (F) LCL-BSTAT4 cells were either nontransduced (NT) or transduced with retroviruses generated with an empty vector or with vectors containing the WT or C115Y mutant versions of IL23R. Cells were either left untreated or treated with kifunensine to inhibit N-glycosylation. Western blotting was performed with antibodies against the V5 tag or GAPDH as a loading control. (G) LCL-BSTAT4 cells from (F) were stained with an anti–IL-23R biotinylated primary antibody and then with avidin–phycoerythrin (PE), and the mean fluorescence intensity (MFI) for the PE signal was quantified by flow cytometry. Error bars represent SEM. (H) LCL-BSTAT4 cells either were left nontransduced (NT) or were transduced with retroviruses generated with an empty vector or vectors containing either V5-tagged WT or Q138X mutant versions of IL12RB2 or V5-tagged WT or C115Y versions of IL23R. The cells were left unstimulated or were stimulated with IL-12 or IFN-α. Cell lysates were prepared, and Western blotting was performed with antibodies against pSTAT4, total STAT4, and GAPDH. (I) LCL-BSTAT4 cells from (H) were left unstimulated or were stimulated with IL-23 or IFN-α as a positive control. Cell lysates were prepared, and Western blotting was performed with antibodies against pSTAT3, total STAT3, and GAPDH. (J) HVS-T cells from a healthy control (C+), an IL-12Rβ1−/− patient, or P1 (IL-12Rβ2 Q138X) were not transduced (NT) or were transduced with a retrovirus generated with an empty vector or a WT allele of IL12RB2 and then stimulated with IL-12 or IFN-α. Western blotting was performed as described in (H). (K) EBV-B cells from a healthy control (C+), an IL-12Rβ1−/− patient, or P4 (IL-23R C115Y) were not transduced (NT) or were transduced with a retrovirus generated with an empty vector or a WT allele of IL23R and then stimulated with IL-23 or IFN-α. Western blotting was performed as described in (I).

  • Fig. 2 IL12RB2 and IL23R are evolving under weak purifying selection, and homozygous complete LOF mutations of these genes occur rarely in the general population.

    (A and B) Graphic representation of all genes of the human genome according to the log10 of their (A) NI or (B) GDI (32)). IL12RB2 (blue), IL23R (red), and IL12RB1 (gray) are each indicated by a vertical dashed line. (C and D) All homozygous variations found in gnomAD for IL12RB2 (C) and IL23R (D) are plotted according to their CADD score (y axis) and minor allele frequency (MAF; x axis). The dashed line indicates the MSC (27) for each gene. (E) LCL-BSTAT4 cells were left nontransduced (NT) or were transduced with retroviruses generated with an empty vector or vectors containing V5-tagged WT or mutant versions of IL12RB2, including the 16 mutations indicated. These IL12RB2 mutations are present in at least one individual in the gnomAD database. Cells were left unstimulated or were stimulated with IL-12 or IFN-α. Cell lysates were prepared, and Western blotting was performed with antibodies against pSTAT4, total STAT4, and GAPDH. (F) LCL-BSTAT4 cells were left nontransduced (NT) or were transduced with retroviruses generated with an empty vector or vectors containing V5-tagged WT or mutant versions of IL23R for the 14 mutations indicated. With the exception of the C115Y mutation found in kindred B, these IL23R mutations are each present in at least one individual in the gnomAD database. Cells were left unstimulated or were stimulated with IL-23 or IFN-α. Cell lysates were prepared, and Western blotting was performed with antibodies against pSTAT3, total STAT3, and GAPDH.

  • Fig. 3 Frequencies and responses of CD4+ T cells from IL-12Rβ1–, IL-12Rβ2–, and IL-23R–deficient individuals to polyclonal stimuli or TH-polarizing cytokines.

    (A) On the left, frequencies of naïve (CCR7+CD45RA+), central memory (CCR7+CD45RA), effector memory (CCR7CD45RA), and T effector memory RA (EMRA) (CCR7CD45RA+) CD4+CD3+ T cells were measured in healthy controls (n = 16), IL-12Rβ2 Q138X patients (n = 2, one measured twice), one IL-23R C115Y patient (technical triplicates), and IL-12Rβ1−/− patients (n = 5). The values shown are the percentage of total CD4+ T cells. On the right, frequencies of four subsets of memory CD4+ T cells (CD3+CD45RA): TH1 (CCR6CCR4CXCR3+), TH1* (CCR6+CCR4CXCR3+), TH17 (CCR6+CCR4+CXCR3), and TH1 (CCR6CCR4+CXCR3) were measured in the same individuals as in the left panel. Error bars indicate the SEM. P values for the comparison of healthy controls with IL-12Rβ1–deficient patients in Mann-Whitney U tests are shown. Formal statistical comparisons including IL-12Rβ2 Q138X and IL-23R C115Y patients were not appropriate because of the extremely small number of individuals (two and one, respectively). (B) Naïve CD4+ T cells from healthy controls, two IL-12Rβ2 Q138X patients (P1 and P2), one IL-23R C115Y patient (P4), and four IL-12Rβ1–deficient patients either were left unpolarized (TH0) or were polarized under TH1 T cell activation/expansion (TAE) beads + IL-12], TH17 conditions (TAE + IL-1/IL-6/IL-21/IL-23/transforming growth factor–β), or TH2 conditions (TAE + IL-4). The production of IFN-γ, IL-17F, and IL-10 was measured with cytometric bead arrays, in cell culture supernatants, after 5 days. (C) Naïve and memory CD4+ T cells from healthy controls, two IL-12Rβ2 Q138X (P1 and P2), one IL-23R C115Y (P4), and four IL-12Rβ1–deficient patients were stimulated with TAE beads, and cytokine production was measured 5 days later. Data for the production of IFN-γ, TNF, and IL-2 (top) and for IL-17A, IL-17F, and IL-10 (bottom) are shown.

  • Fig. 4 Specific defects in the IL-12– and IL-23–dependent generation of an IFN-γ immune response in patients with novel homozygous mutations of IL12RB2 or IL23R.

    (A and B) Multiple CCR6+ or CCR6 memory CD4+ T cell lines were generated by the polyclonal stimulation of sorted peripheral blood subsets from healthy controls (n = 4), one IL-12Rβ2 Q138X patient, one IL-23R C115Y patient, and three IL-12Rβ1–deficient patients. Lines were screened for reactivity with peptide pools covering antigens from BCG (top) and MTB (bottom). BCG- and MTB-reactive CD4+CCR6+ T cell lines from each individual were selected, and the cytokines accumulating in the culture supernatant were determined with a Luminex machine. Each dot on the graph corresponds to a value for a single antigen-reactive T cell line. CCR6+ T cell lines are shown as closed circles, and CCR6 cell lines are shown as open circles. (C and D) Microarray heat map of isolated B cells, CD4+ T cells, CD8+ T cells, γδ+ T cells, and NK cells stimulated with IL-12 (C) or IL-23 (D) for 6 hours. The data shown are the fold induction relative to nonstimulated (ns) cells. The most commonly up-regulated gene, IFNG, is highlighted in the bottom right corner of each heat map. (E) IFNG induction by isolated B cells, CD4+ T cells, CD8+ T cells, γδT cells, and NK cells from five healthy controls, upon stimulation with IL-12 or IL-23 for 6 hours, was assessed by qPCR, and the data were normalized relative to nonstimulated cells. (F) IFN-γ levels in the supernatants from the cells used in (E) were analyzed by ELISA and represented as a fold change relative to nonstimulated cells. (G and H) Sorted MAIT cells (G) or NKT cells (H) (>95% pure) were left unstimulated or stimulated with rhIL-12 (20 ng/ml) or rhIL-23 (100 ng/ml) for 6 hours. Cell culture supernatants were harvested and used for IFN-γ determination in a multiplex cytokine assay. (I) NK cells, ILC1, or ILC2 were sorted, by fluorescence-activated cell sorter (FACS), from blood samples from healthy donors and cultured in the presence of the indicated cytokines for 24 hours. Total ILCs were gated on viable CD45+Lin (CD3CD4CD5TCRαβTCRγδCD14CD19) CD7+ cells. NK cells were identified as CD56bright and CD56dim, ILC2 as CD56CD127+CRTH2+ and ILC1 as CD56CD127+CD117CRTH2. IFN-γ levels were determined by intracellular staining. ILC3 were sorted by FACS from the tonsillar tissues of healthy donors by gating on viable CD45+ LinCD7+CD117+NKp44+ cells. ILC3 was cultured for 4 days in the presence of the indicated cytokines, and IFN-γ was then determined by intracellular staining.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/30/eaau6759/DC1

    Ethics statement

    Case reports

    Supplementary Materials and Methods

    Fig. S1. Clinical and genetic features of kindreds with AR IL-12Rβ2 or IL-23R deficiency.

    Fig. S2. IL12RB2 Q138X and IL23R C115Y mutations abolish the response of the encoded receptor to its cognate cytokine ligand.

    Fig. S3. Population genetics for IL12RB1, IL12RB2, and IL23R.

    Fig. S4. Immunophenotype of IL-12Rβ2–, IL-12Rβ1–, and IL-23R–deficient patients.

    Fig. S5. Flow cytometry gating.

    Fig. S6. Nonantigen-specific cytokine production, and antigen-specific proliferation, precursor frequency, and cytokine production by memory CD4+ T cells from healthy controls and IL-12Rβ2–, IL-23R–, and IL-12Rβ1–deficient patients.

    Fig. S7. Cytokine receptor expression and responses of MAIT cells and NKT cells to stimulation with IL-12 or IL-23.

    Table S1. Estimated proportions of deleterious and nondeleterious amino acid variants in IL12RB1, IL12RB2, and IL23R.

    Table S2. Relative levels of IFN-γ production in response to IL-12 versus IL-23 in different cell subsets.

    Table S3. Raw data used to generate dot plots, heat maps, and bar graphs.

    References (5669)

  • Supplementary Materials

    The PDF file includes:

    • Ethics statement
    • Case reports
    • Supplementary Materials and Methods
    • Fig. S1. Clinical and genetic features of kindreds with AR IL-12Rβ2 or IL-23R deficiency.
    • Fig. S2. IL12RB2 Q138X and IL23R C115Y mutations abolish the response of the encoded receptor to its cognate cytokine ligand.
    • Fig. S3. Population genetics for IL12RB1, IL12RB2, and IL23R.
    • Fig. S4. Immunophenotype of IL-12Rβ2–, IL-12Rβ1–, and IL-23R–deficient patients.
    • Fig. S5. Flow cytometry gating.
    • Fig. S6. Nonantigen-specific cytokine production, and antigen-specific proliferation, precursor frequency, and cytokine production by memory CD4+ T cells from healthy controls and IL-12Rβ2–, IL-23R–, and IL-12Rβ1–deficient patients.
    • Fig. S7. Cytokine receptor expression and responses of MAIT cells and NKT cells to stimulation with IL-12 or IL-23.
    • Table S1. Estimated proportions of deleterious and nondeleterious amino acid variants in IL12RB1, IL12RB2, and IL23R.
    • Table S2. Relative levels of IFN-γ production in response to IL-12 versus IL-23 in different cell subsets.
    • Legend for table S3
    • References (5669)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S3 (Microsoft Excel format). Raw data used to generate dot plots, heat maps, and bar graphs.

    Files in this Data Supplement:

Stay Connected to Science Immunology

Navigate This Article