Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Dec 21;3(30):eaau6759.
doi: 10.1126/sciimmunol.aau6759.

Human IFN-γ Immunity to Mycobacteria Is Governed by Both IL-12 and IL-23

Affiliations
Free PMC article

Human IFN-γ Immunity to Mycobacteria Is Governed by Both IL-12 and IL-23

Rubén Martínez-Barricarte et al. Sci Immunol. .
Free PMC article

Abstract

Hundreds of patients with autosomal recessive, complete IL-12p40 or IL-12Rβ1 deficiency have been diagnosed over the last 20 years. They typically suffer from invasive mycobacteriosis and, occasionally, from mucocutaneous candidiasis. Susceptibility to these infections is thought to be due to impairments of IL-12-dependent IFN-γ immunity and IL-23-dependent IL-17A/IL-17F immunity, respectively. We report here patients with autosomal recessive, complete IL-12Rβ2 or IL-23R deficiency, lacking responses to IL-12 or IL-23 only, all of whom, unexpectedly, display mycobacteriosis without candidiasis. We show that αβ T, γδ T, B, NK, ILC1, and ILC2 cells from healthy donors preferentially produce IFN-γ in response to IL-12, whereas NKT cells and MAIT cells preferentially produce IFN-γ in response to IL-23. We also show that the development of IFN-γ-producing CD4+ T cells, including, in particular, mycobacterium-specific TH1* cells (CD45RA-CCR6+), is dependent on both IL-12 and IL-23. Last, we show that IL12RB1, IL12RB2, and IL23R have similar frequencies of deleterious variants in the general population. The comparative rarity of symptomatic patients with IL-12Rβ2 or IL-23R deficiency, relative to IL-12Rβ1 deficiency, is, therefore, due to lower clinical penetrance. There are fewer symptomatic IL-23R- and IL-12Rβ2-deficient than IL-12Rβ1-deficient patients, not because these genetic disorders are rarer, but because the isolated absence of IL-12 or IL-23 is, in part, compensated by the other cytokine for the production of IFN-γ, thereby providing some protection against mycobacteria. These experiments of nature show that human IL-12 and IL-23 are both required for optimal IFN-γ-dependent immunity to mycobacteria, both individually and much more so cooperatively.

Figures

Figure 1:
Figure 1:. Identification of homozygous complete loss-of-function IL-12Rβ2 Q138X and IL-23R C115Y mutations in families with MSMD.
A,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, 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 cells were either non-transfected (NT) 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 GAPDH as a loading control. F) LCL-BSTAT4 cells were either non-transduced (NT), or transduced with retroviruses generated with an empty vector (EV) 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, then with avidin-PE, and the mean fluorescence intensity (MFI) for the PE signal was quantified by flow cytometry. H) LCL-BSTAT4 cells were either left non-transduced (NT), or were transduced with retroviruses generated with an empty vector (EV) or vectors containing V5-tagged WT or Q138X mutant versions of IL12RB2, or 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-12Rβ−/− patient or P1 (IL-12Rβ2 Q138X) were not transduced (NT), or were transduced with a retrovirus generated with an empty vector (EV) 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 (EV) or a WT allele of IL23R and then stimulated with IL-23 or IFN- α. Western blotting was performed as described in (I).
Figure 2:
Figure 2:. IL12RB2 and IL23R are evolving under weak purifying selection, and homozygous complete loss-of-function mutations of these genes occur rarely in the general population.
A,B) Graphic representation of all genes of the human genome according to the log10 of their (A) neutrality index (NI) or (B) gene damage index (GDI, (31)). IL12RB2 (blue), IL23R (red), and IL12RB1 (grey) are each indicated by a dashed vertical line. C, 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 mutation significance cutoff (MSC, (26)) for each gene. E) LCL- BSTAT4 cells were left non-transduced (NT), or were transduced with retroviruses generated with an empty vector (EV) 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 non-transduced (NT), or were transduced with retroviruses generated with an empty vector (EV) 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.
Figure 3:
Figure 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 naive (CCR7+CD45RA+), central memory (CCR7+CD45RA), effector memory (CCR7CD45RA) and TEMRA (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 rL-12Rβ1−/− patients (n=5). The values shown are the percentage of total CD4+ T cells. On the right, frequencies of four subsets of CD4+ memory T cells (CD3+CD45RA): Th1 (CCR6CCR4CXCR3+), Th1* (CCR6+CCR4CXCR3+), Th17 (CCR6+CCR4+CXCR3) and Th2 (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, due to the extremely small number of individuals (2 and 1, 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 were either left unpolarized (Th0) or were polarized under Th1 conditions (TAE+IL-12), Th17 conditions (TAE+IL-1/IL-6/IL-21/IL-23/TGF-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 are shown in the upper panels and for IL-17A, IL-17F and IL-10 in the lower panels.
Figure 4:
Figure 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, 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 (upper panel) and MTB (lower panel). 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, D) Microarray heat map of isolated B, CD4+ T, CD8+ T, γδ+ T and NK cells stimulated with IL-12 (C) or IL-23 (D) for 6 h. The data shown are the fold-induction relative to non-stimulated (n.s.) cells. The most commonly upregulated gene, IFNG, is highlighted in the lower right corner of each heat map. E) IFNG induction by isolated B, CD4+ T, CD8+ T, γδT, and NK cells from 5 healthy controls, upon stimulation with IL-12 or IL-23 for 6 h, was assessed by qPCR, and the data were normalized relative to n.s. 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 n.s. cells. G, 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 h. 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 FACS, from blood samples from healthy donors and cultured in the presence of the indicated cytokines for 24 h. 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 were cultured for 4 days in the presence of the indicated cytokines, and IFN-γ was then determined by intracellular staining.

Similar articles

See all similar articles

Cited by 21 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback