Research ArticleTUBERCULOSIS

Tuberculosis and impaired IL-23–dependent IFN-γ immunity in humans homozygous for a common TYK2 missense variant

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Science Immunology  21 Dec 2018:
Vol. 3, Issue 30, eaau8714
DOI: 10.1126/sciimmunol.aau8714
  • Fig. 1 Familial segregation and clinical information for patients homozygous for TYK2 P1104A.

    (A) Schematic diagram of the TYK2 protein with its various domains (FERM, SH2, pseudokinase, and tyrosine kinase). The positions of the previously reported TYK2 mutations resulting in premature STOP codons are indicated in red. The positions of the I684S and P1104A polymorphisms are indicated in blue and green, respectively. (B) Pedigrees of the 10 TYK2-deficient families. Each generation is designated by a Roman numeral (I–II), and each individual by an Arabic numeral. The double lines connecting the parents indicate consanguinity based on interview and/or a homozygosity rate of >4% estimated from the exome data. Solid shapes indicate disease status. Individuals whose genetic status could not be determined are indicated by “E?”, and “m” indicates a TYK2 P1104A allele. (C) Summary table of clinical details and origin of the patients associated with the MAF in the country of origin. The incidence of tuberculosis (TB) in the country of residence is also mentioned. MAC indicates Mycobacterium avium complex. (D) Summary of WES, indicating the numbers of individuals with tuberculosis or MSMD and of controls carrying the I684S or P1104A variant of TYK2 in the homozygous state, and the associated P value and OR. (E) Distributions of the current allele frequencies of variants that segregated 4000 years ago at frequencies similar to those of the P1104A and I684S TYK2, M694V MEFV, and C282Y HFE variants. The red vertical lines indicate the current frequency of the four variants of interest. Colored bars indicate the distribution of current allele frequency, in the 1000 Genomes Project, for variants with frequencies in ancient European human DNA similar to those of the four candidate variants (52). Black lines indicate the distribution of simulated frequencies, in the present generation, for alleles with a past frequency similar to that of the four candidate variants, with propagation over 160 generations (corresponding to a period of ~4000 years) under the Wright-Fisher neutral model. For instance, for the P1104A allele, which had a frequency of ~9% in ancient Europeans, colored bars indicate the observed distribution of current frequencies for the 31,276 variants with a frequency of 8 to 10% 4000 years ago. The black lines indicate the distribution of frequencies for 100,000 simulated alleles obtained after 160 generations under the Wright-Fisher neutral model.

  • Fig. 2 Cellular responses to IFN-α, IL-12

    and IL-23 in transduced EBV-B and HVS-T cells. TYK2-deficient EBV-B and HVS-T cells were transduced with a retrovirus generated with an empty vector (EV), or vectors encoding WT TYK2, or the P1104A, I684S, or K930R TYK2 alleles. (A) Levels of TYK2 in transduced EBV-B (left) and HVS-T (right) cells, as determined by Western blotting. (B) Levels of IL-12Rβ1 and IFN-αR1 in transduced EBV-B (left) and HVS-T (right) cells, as determined by flow cytometry. ***P < 0.001, two-tailed Student’s t test. Error bars indicate SEM. (C, D, and F) Phosphorylation of JAKs and STATs in unstimulated (−) transduced EBV-B or HVS-T cells or in these cells after stimulation (+) with IFN-α (C) (pTYK2, pJAK1, and pSTAT1), IL-12 (D) (pTYK2, pJAK2, pSTAT1, and pSTAT4), and IL-23 (F) (pTYK2, pJAK2, pSTAT3, and pSTAT1), as assessed by Western blotting with specific antibodies recognizing phospho-TYK2, phospho-JAK1, phospho-JAK2, phospho-STAT1, phospho-STAT4, and phospho-STAT3. MW, molecular weight. (E) Phosphorylation of STAT4 in response to IFN-α and IL-12, as determined by flow cytometry in HVS-transduced T cells and expression as mean fluorescence intensity (MFI). **P < 0.01, ***P < 0.001, two-tailed Student’s t test. ns, not significant. (G) IFN-β response of U1A (left) and MEF (right) cells, both lacking TYK2, after transduction with the indicated human and mouse TYK2 alleles, respectively, or with empty vector control, as measured in an IFN-β–induced antiviral activity assay (see Materials and Methods). A unique dose is shown: an IFN-β dose of 0.01 ng/ml for human cells and 1 IU/ml for mouse cells.

  • Fig. 3 Cellular responses to IFN-

    α and IL-12 in cell lines from patients. (A) TYK2 levels in EBV-B cells from two controls, two TYK2-deficient patients, two patients homozygous for TYK2 P1104A, two patients homozygous for TYK2 I684S, and a patient compound heterozygous for the P1104A/I684S TYK2 alleles, as assessed by Western blotting. (B) Levels of IL-12Rβ1 in EBV-B cells and HVS-T cells and of IFN-αR1 in EBV-B cells from controls, TYK2-deficient patients, patients homozygous for TYK2 P1104A, patients homozygous for TYK2 I684S, and a patient compound heterozygous for P1104A/I684S TYK2 alleles, as assessed by flow cytometry. *P < 0.05, **P < 0.01, two-tailed Student’s t test. (C and D) Phosphorylation of JAKs and STATs in EBV-B or HVS-T cells of the indicated TYK2 genotypes after stimulation with IFN-α (C) (pTYK2, pJAK1, pSTAT1, and pSTAT3) or IL-12 (D) (pTYK2, pJAK2, and pSTAT4), as determined by Western blotting.

  • Fig. 4 Cellular responses to IL-23 and IFN-

    α in cell lines from patients. (A) Phosphorylation of JAKs and STATs in EBV-B cells carrying the indicated TYK2 genotypes after stimulation with IL-23 (pTYK2, pJAK2, pSTAT3, and pSTAT1). (B) Phosphorylation of STAT3 after stimulation with IFN-α or IL-23, in HVS-T cells of the indicated genotypes, as assessed by flow cytometry. (C) Expression patterns on RNA-seq of EBV-B cells stimulated with IFN-α. The heat map represents the fold change (FC) difference in expression before and after stimulation on a log2 scale. Red blocks represent up-regulated genes, and blue blocks represent down-regulated genes. The genes up-regulated with an FC of ≥2.5, i.e., log2(FC) ≥ 1.3, in the group of controls are shown. (D) Relative levels of SOCS3 expression in EBV-B cells after IL-23 stimulation. (E) Percentage of cell death for primary fibroblasts of the indicated genotype after VSV infection at various MOIs, with and without IFN-β pretreatment.

  • Fig. 5 Molecular mechanisms of impaired response to IL-23 by TYK2 P1104A.

    (A) In vitro kinase assay performed in the presence or absence of added ATP on TYK2 immunopurified from human TYK2-deficient cells (U1A) stably reconstituted with either TYK2 WT or TYK2 P1104A. RecSTAT3 (top) or recSTAT1 (bottom) was added to the reaction mixture. The products of the reaction were analyzed by immunoblotting with antibodies specific to the two activation loop tyrosine residues of TYK2 (Tyr1054–1055), phospho-STAT1 (Tyr701), or phospho-STAT3 (Tyr705). (B) Fold change in Renilla luciferase (RLU) after stimulation with IL-23 (left) or IL-12 (right) in TYK2−/− cells stably reconstituted with IL-12Rβ1 and IL-23R (left) or IL-12Rβ1 and IL-12Rβ2 (right). Cells were left untransfected, or were transfected with JAK2 and TYK2 fused to Rluc fragments, for the detection of interactions after stimulation. The TYK2 used was either WT or P1104A. (C) Phosphorylation of JAK2, TYK2, STAT1, and STAT3 after stimulation with IL-12 and IL-23 in TYK2−/− and JAK2−/− fibrosarcoma cells reconstituted with IL-12Rβ1, IL-12Rβ2, and IL-23R. TYK2−/− cells were transfected with WT, P1104A, or K930R TYK2, and JAK2−/− cells were transfected with WT, P1057A, or K882E JAK2.

  • Fig. 6 Analysis of primary cells from patients.

    (A) ELISA analysis of IFN-γ levels in whole blood after stimulation with BCG, or BCG plus IL-12, in travel controls, TYK2-deficient and IL-12Rβ1–deficient patients, and patients homozygous for the P1104A or I684S TYK2 alleles. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t tests. (B) Production of IFN-γ from PBMCs stimulated with BCG, BCG plus IL-12, BCG plus IL-23, or PMA plus ionomycin (PMA) in healthy controls, homozygous TYK2 P1104A patients, hyper-IgE patients with heterozygous STAT3 mutations (STAT3-DN), and patients with complete TYK2 and IL-12Rβ1 deficiencies, as determined by ELISA. (C) Percentages of IL-17A–, IL-17F–, and IFN-γ–positive CD4+ T cells after the stimulation of PBMCs from healthy controls and patients homozygous for TYK2 P1104A with PMA plus ionomycin. (D) In vitro differentiation of naïve CD4+ T cells from healthy controls, patients homozygous for P1104A TYK2 alleles, and patients with TYK2 deficiency, after culture under TH17 (with IL-23) or TH1 (with IL-12) polarizing conditions, as determined by assessments of the induction of IL-17A/F and IFN-γ secretion, respectively. (E) Production of IFN-γ, IL-17A, IL-17F, and IL-22 by naïve and memory CD4+ T cells from healthy controls, patients homozygous for P1104A TYK2, and TYK2-deficient patients, stimulated with TAE beads for 5 days.

  • Fig. 7 Schematic representation of TYK2-dependent signaling pathways.

    The cytokines, receptors, and JAK and STAT complexes formed are indicated. A summary of the functionality of each pathway is provided for the various genotypes: TYK2 WT/WT, TYK2−/−, TYK2 I684S/I684S, and TYK2 P1104A/P1104A. The main STAT-containing complexes are shown. Other complexes include STAT1/STAT1 in response to IL-10, IL-12, IL-23, and IFN-α/β and STAT3/STAT3 and STAT4/STAT4 in response to IFN-α/β. The symbol +++ indicates that the pathway is functional and optimal (corresponding to WT TYK2). The symbol ++ indicates that the function of the pathway is decreased without overt clinical implications. The symbol + means that the function of the pathway is impaired, but not completely abolished because of TYK2-independent residual signaling, and can bear clinical consequences (except for IL-10). The clinical phenotypes of individuals homozygous for the WT, P1104A, and I684S TYK2 alleles are indicated on the right.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/30/eaau8714/DC1

    Materials and Methods

    Case Reports

    Fig. S1. Population and medical genetics.

    Fig. S2. Response to IFN-α/β, IL-10, IL-12, and IL-23 in TYK2-deficient cell lines transduced with different TYK2 alleles.

    Fig. S3. Response to IFN-α/β in cell lines with different TYK2 genotypes.

    Fig. S4. Response to IL-12 and IL-10 in cell lines with different TYK2 genotypes.

    Fig. S5. Response to IL-23 in cell lines with different TYK2 genotypes.

    Fig. S6. Induction of target genes after IL-10, IFN-α, and IL-23 stimulation in cell lines with different TYK2 genotypes.

    Fig. S7. Auto- and transphosphorylation of P1104A TYK2 and P1057A JAK2.

    Fig. S8. Analysis of leukocytes with different TYK2 genotypes.

    Table S1. Summary of the TYK2 genotypes among the different cohorts of patients and healthy individuals.

    Table S2. Raw data used to generate dot plots and bar graphs.

    References (9496)

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Case Reports
    • Fig. S1. Population and medical genetics.
    • Fig. S2. Response to IFN-α/β, IL-10, IL-12, and IL-23 in TYK2-deficient cell lines transduced with different TYK2 alleles.
    • Fig. S3. Response to IFN-α/β in cell lines with different TYK2 genotypes.
    • Fig. S4. Response to IL-12 and IL-10 in cell lines with different TYK2 genotypes.
    • Fig. S5. Response to IL-23 in cell lines with different TYK2 genotypes.
    • Fig. S6. Induction of target genes after IL-10, IFN-α, and IL-23 stimulation in cell lines with different TYK2 genotypes.
    • Fig. S7. Auto- and transphosphorylation of P1104A TYK2 and P1057A JAK2.
    • Fig. S8. Analysis of leukocytes with different TYK2 genotypes.
    • Table S1. Summary of the TYK2 genotypes among the different cohorts of patients and healthy individuals.
    • References (9496)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Raw data used to generate dot plots and bar graphs.

    Files in this Data Supplement:

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