TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9

Abstract

Toll-like receptors (TLRs) have a crucial role in the recognition of pathogens and initiation of immune responses^1-3. Here we show that a previously uncharacterized protein encoded by CXorf21-a gene that is associated with systemic lupus erythematosus^4,5-interacts with the endolysosomal transporter SLC15A4, an essential but poorly understood component of the endolysosomal TLR machinery also linked to autoimmune disease^4,6-9. Loss of this type-I-interferon-inducible protein, which we refer to as 'TLR adaptor interacting with SLC15A4 on the lysosome' (TASL), abrogated responses to endolysosomal TLR agonists in both primary and transformed human immune cells. Deletion of SLC15A4 or TASL specifically impaired the activation of the IRF pathway without affecting NF-κB and MAPK signalling, which indicates that ligand recognition and TLR engagement in the endolysosome occurred normally. Extensive mutagenesis of TASL demonstrated that its localization and function relies on the interaction with SLC15A4. TASL contains a conserved pLxIS motif (in which p denotes a hydrophilic residue and x denotes any residue) that mediates the recruitment and activation of IRF5. This finding shows that TASL is an innate immune adaptor for TLR7, TLR8 and TLR9 signalling, revealing a clear mechanistic analogy with the IRF3 adaptors STING, MAVS and TRIF^10,11. The identification of TASL as the component that links endolysosomal TLRs to the IRF5 transcription factor via SLC15A4 provides a mechanistic explanation for the involvement of these proteins in systemic lupus erythematosus^12-14.

Extended Data Figure 1. Subcellular localization of tagged SLC15A4 and related constructs in THP1 cells.

(a) Gene expression levels of SLC15A1-4 in THP1 cells from (b) Domain organization of SLC15A4 protein. TM: transmembrane domain. (c,d) Confocal microscopy of indicated THP1 cells. Red: anti-HA, green: anti-Lamp1, blue: DAPI. Scale bar: 10 μm. (e) Lysates from indicated THP1 cells untreated or treated with PNGase F were analyzed by immunoblotting. (c-e) Data representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Figure 2. TASL is an immune cell-restricted protein conserved in vertebrates.

(a) Multiple sequence alignment of TASL protein from representative vertebrate species. UniProt entry names: CX021_HUMAN, H2QYF9_PANTR, CX021_BOVIN, CX021_MOUSE, H0Z9M3_TAEGU, A0A1L1RS25_CHICK, A0A1U7RX84_ALLSI, G1KG99_ANOCA, F7BWY0_XENTR, W5NMP6_LEPOC, I3KXE0_ORENI. Boxes above the alignment indicate consensus prediction from JPred4. Red: helix, yellow: β sheet. (b, c) Expression levels of SLC15A4 and TASL in (b) primary human cells as measured by CAGE by FANTOM5 and in (c) human cancer cell lines measured by RNA-Seq from. Circles represent individual samples, in boxplots, bars indicate median, boxes the first–third quartiles. Upper whisker extends from hinge to largest value no further than 1.5 * IQR (inter-quartile range) from the hinge. Lower from hinge to smallest value at most 1.5 * IQR of the hinge.

Extended Data Figure 3. Type I interferon-inducible TASL forms a complex with SLC15A4.

(a) Normalized mRNA expression of SLC15A4 and TASL relative to GAPDH in THP1 cells treated as indicated. Data show mean (n=3 technical replicates). (b) Immunoblots of lysates of THP1 cells stimulated with interferon β or γ (20 ng/ml, 16h) treated with PNGase F as indicated. (c,e) Lysates from (c) KBM7 cells transduced or (e) HEK293T cells transiently transfected as indicated were subjected to HA-immunoprecipitation. Immunoprecipitates (IP) and whole cell extracts (WCE) were analyzed by immunoblotting. (d) Overview of deletion mutants used in Fig. 1g. (f) Lysates from indicated THP1 cells were subjected to HA-immunoprecipitation and treated or not with λ phosphatase. IP and WCE were analyzed by immunoblotting. (g) Immunoblots of indicated THP1 cells treated with PNGase F. (h) Multiple sequence alignment of human and murine SLC15A3 and SLC15A4. UniProt entry names: S15A3_HUMAN, S15A3_MOUSE, S15A4_HUMAN, S15A4_MOUSE. (a-c,e-g) Data representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Figure 4. SLC15A4 controls TASL protein abundance and subcellular localization.

(a,b) (a) Flow cytometry and (b) immunoblot of indicated THP1 cells. (c) Confocal live microscopy of indicated THP1 cells. Green: TASL-GFP, Red: Lysotracker, Blue: Hoechst33342. Scale bar: 10 μm. (d) Upper panel: Confocal microscopy of indicated formaldehyde-fixed THP1 cells. Green: TASL-GFP, Red: HA, Blue: DAPI. Scale bar: 10 μm. Lower panel: profiles of signal intensity of TASL-GFP (green) and HA (red) along the red lines shown in microscopy images (upper panel). (a-d) Data representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Figure 5. TASL mirrors SLC15A4 requirement for TLR7/8 activation.

(a) Fraction of R848-induced genes affected by SLC15A4 and TASL knockout, related to Fig. 2b. (b) Upset plot representing number of R848-induced genes commonly affected by indicated sgRNAs, related to Fig. 2b. (c) TASL gene expression levels in indicated THP1 cells, related to Fig. 2b. Bar graphs shown mean (n=3 biological replicates), error bars show 95 % confidence interval of mean. (d) Flow cytometry of PD-L1 surface expression in indicated unstimulated (ns) or R848-stimulated (5 μg/ml, 24h) THP1 cells. (e) Immunoblots of indicated THP1 DUAL cells. Lysates treated with PNGase F as indicated. (f-i,k) Indicated THP1 DUAL cells were (co-)treated for 24 hours with R848 (5 μg/ml), CL075 (5 μg/ml), ssRNA40/LyoVec™ (5 μg/ml) or inactive control ssRNA41/LyoVec™ (5 μg/ml), C12-iE-DAP (5 μg/ml), MDP (10 μg/ml), murabutide (10 μg/ml), Pam3CSK4 (0.1 μg/ml), Flagellin (0.1 μg/ml), cGAMP (3 μg/ml) or interferon β (20 ng/ml). (h) CRISPR/Cas9 editing efficiency (%) estimated by TIDE. (j) Indicated THP1 DUAL cells were primed or not with interferon γ (0.1 μg/ml) for 24h, washed and stimulated or not with MDP (10 μg/ml, 24h). (f-k) Supernatants were analyzed for ISRE and NF-κB reporter activity. Mean ± s.d. (n=3 biological replicates). (l) Relative mRNA expression of SLC15A4, TASL or MYD88 in siRNA-transfected CD14⁺ monocytes in comparison to control (siCTRL). Data represent mean ± s.d. from six (MYD88, n=6) or seven (SLC15A4, TASL, n=7) individual donors. (d-k) Data representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Figure 6. TASL and SLC15A4 deficiency impairs endosomal TLR-mediated signaling downstream of receptor engagement.

(a) Immunoblots of lysates of THP1 cells treated with PNGase F as indicated. (b,c) Cytokine production of indicated THP1 cells unstimulated or stimulated with CpG-A, CpG-B (5 μM) or R848 (5 μg/ml) for 24h. Data show mean ± s.d. of biological replicates (TNF, CCL2: n=3; IFN β: n=2). (d) Immunoblots of indicated THP1 cells stimulated or not with interferon γ (0.1 μg/ml, 16h). (e) Immunoblots of indicated THP1 cells. (f) Indicated THP1::TLR9 cells treated with FITC-labeled CpG-A or CpG-B (1 μM, 0-120 min.) were analyzed by flow cytometry. (g) Representative flow cytometry scatter plots of phagocytosis assays. Differentiated THP1 cells, treated or not with Bafilomycin A1, were incubated with dual-colored opsonized beads. Using intensities of pH-insensitive (YG) and pH-sensitive (pHrodo-Red, signal increases with decrease in pH) dyes, cells are divided into phagocytosis negative (PhagoNeg: double-negative), cells that have undergone phagocytosis and phagosome acidification (PhagoLate: double-positive) and early phagocytic cells (PhagoEarly: YG and low pHrodo-Red signal). The marginal intensity distributions are displayed on the sides of the plot. (h) Bar graphs show mean ± s.d. (n=3 biological replicates) of fractions described in (g). (i,j) Indicated THP1 cells were subjected to phagocytosis assays. (i) Bar graphs show mean ± s.d. (n=3 biological replicates) of fractions described in (g). (j) Bar graphs represent mean MFI ± s.d. (n=3 biological replicates) of the pHrodo-Red signal acquired in the MFI gate shown in (g) focusing on cells having taken up 1 to 3 beads/cell. (k) Flow diagram for quantification of Lysosensor™ Green intensities in lysosomal compartments by microscopy. Box plots show intensity of Lysosensor signal on Lysotracker positive lysosomes, as measured by imaging in the indicated THP1 cells. Bars indicate median, boxes the first–third quartiles. Upper whisker extends from hinge to largest value no further than 1.5 * IQR (inter-quartile range) from the hinge. Lower from hinge to smallest value at most 1.5 * IQR of the hinge. Outliers are shown as circles. sgRen: n=2432, sgSLC15A4-1: n=1721, sgSLC15A4-2: n=1981, sgTASL-1: n=2378, sgTASL-2: n=2627 quantified speckles. (l) Immunoblots of indicated THP1 cells stimulated with R848 (5 μg/ml, 0-180 min.). (a-k) Data representative of (a-f,k,l) two or (g-j) three independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Figure 7. TASL/SLC15A4 loss mirrors IRF5-deficiency in perturbing endosomal TLR responses.

(a,d,e) THP1 DUAL cells were (co-)treated for 24 hours with R848 (5 μg/ml), LPS (0.1 μg/ml), Pam3CSK4 (0.1 μg/ml), cGAMP (3 μg/ml), Flagellin (0.1 μg/ml) or MDP (10 μg/ml) as indicated. Supernatants were analyzed for ISRE and NF-κB reporter activity. (b,c) Immunoblots of indicated (b) THP1 DUAL or (c) THP1::TLR9 cells. (f) TNF production of indicated THP1::TLR9 cells stimulated with CpG-B (2 μM, 24h). (g) Immunoblots of indicated THP1 cells stimulated or not with R848 (5 μg/ml, 3h). (h) Upset plot representing number of CpG-B-induced genes (2 μM, 6h; DESeq2 adjusted p-value < 0.05, n=3 biological replicates) in comparison to control (sgRen) commonly affected by indicated sgRNAs. No gene was significantly affected by sgIRF7-1. (i) PCA plot of transcriptional profiles of untreated and CpG-B-treated (2 μM, 6h) THP1::TLR9 cells (n=3 biological replicates) shown in Fig. 3d. (j) Heatmap representing 20 most induced genes by CpG-B in control THP1::TLR9 cells and not affected by neither SLC15A4, TASL nor IRF5 knockout, related to Fig. 3d,e. (k) Transcription factor enrichment analysis (two-sided Fisher’s Exact Test, p-value adjusted for multiple testing) of genes upregulated upon CpG-B treatment in control THP1 cells specifically affected (DESeq2 adjusted p-value < 0.05, n=3 biological replicates) (left panel) or not (right panel) by SLC15A4 and TASL knockout, related to Fig. 3d,e. Background sets are defined as all genes upregulated by CpG-B treatment or all expressed genes (counts per million > 1) respectively. (a,d-f) Data show mean ± s.d. (n=3 biological replicates). (a-g) Data representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Figure 8. SLC15A4/TASL complex is required for IRF5-dependent signaling in human pDC CAL-1 cells.

(a-c) Immunoblots of lysates of CAL-1 cells, PNGase F treatment as indicated. (d) Immunoblots of CAL-1 or THP1::TLR9 cells stimulated with R848 (5 μg/ml), CpG-B (5 μM) or cGAMP (3 μg/ml) as indicated. (e,f) Cytokine production of indicated CAL-1 cells stimulated with R848 (5 μg/ml) or imiquimod (IMQ, 5 μg/ml) for 24h. (f) CRISPR/Cas9 editing efficiency (%) estimated by TIDE. (g) Immunoblots of CAL-1 cells stimulated with CpG-B (5 μM, 0-180 min.) as indicated. (h) Lysates from indicated CAL-1 cells were subjected to HA-immunoprecipitation and analyzed by immunoblotting. (i) TNF production of cells described in (h) upon stimulation with R848 (5 μg/ml, 24h). (j) Immunoblots show expression levels (WCE) and HA-immunoprecipitates (IP) from indicated THP1 cells. Bar graphs represent TNF production upon R848 stimulation (5 μg/ml, 24h). (e,f,i,j) Bar graphs show mean ± s.d. (n=3 biological replicates). (a-j) Data representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Figure 9. Mutagenesis of TASL identifies functional elements and reveals a pLXIS motif required for IRF5 activation.

(a) Overview of mutants used in Fig. 4b, changes to alanine indicated by red circles. (b,f) Immunoblots of indicated THP1 cells. Bar graphs represent TNF levels following R848 stimulation (5 μg/ml, 24h). (f) Dashed line indicates cropping of unrelated lanes from the same blots. (c) Immunoblots of indicated reconstituted TASL-deficient THP1 cells. (d) Normal expression levels, but reduced detection by anti-TASL antibodies of TASL mutants targeting aa 261-277. Immunoblots of HEK293T cells transiently transfected with indicated cDNAs. (e) Lysates from indicated THP1 cells were subjected to immunoprecipitation (IP). IP and whole cell extracts (WCE) were analyzed by immunoblotting. (g) Abundance of indicated proteins determined by MS in V5-immunoprecipitates from THP1::TLR9 cells stimulated with CpG-B (5 μM, 2h) as indicated, related to Fig. 4g. Three biological replicates are shown. (h) Crystal structures of IRF3 bound to phosphorylated pLxIS-containing peptides from STING (pink, PDB ID 5JEJ), MAVS (green, PDB ID 5JEK) and TRIF (blue, PDB ID 5JEL). Residues in STING peptides shown as sticks. (i) Superposition of peptide-bound IRF3 (PDB ID 5JEJ) and dimeric IRF5 (one monomer shown, PDB ID 3DSH), showing highly similar folds. (j) Model of phosphomimetic pLxIS-containing peptide from TASL (pmTASL, ISTPSLHIDQYSNV, yellow) bound to IRF5. Residues corresponding to LxIS motif shown as sticks. (k) Comparison of binding mode of pLIxS-containing peptides to IRF proteins. Only IRF5 is shown for clarity. (l) Immunoblots of indicated THP1 cells unstimulated or stimulated with R848 (5 μg/ml, 2h). (m) TNF production of cells described in (l) stimulated with R848 (5 μg/ml, 24h). (b,f,m) Bar graphs show mean ± s.d. (n=3 biological replicates). (b-f,l,m) Data are representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Figure 10. IKKβ is required for TASL-dependent IRF5 activation.

(a) Immunoblots of HEK293T cells transfected as indicated. (b) THP1 DUAL cells were pre-treated for 30 min. with DMSO or inhibitors as indicated and stimulated with R848 (5 μg/ml, 24h). Supernatants were analyzed for ISRE and NF-κB reporter activity and normalized to the respective R848-only treated conditions. Three biological replicates are shown (n=3). (c) Immunoblots of THP1::TLR9 cells pre-treated for 30 min. with DMSO or inhibitors (5 μM) and stimulated with CpG-B (5 μM, 4h) as indicated. (d) Lysates from THP1::TLR9 cells pre-treated (30 min.) with DMSO or inhibitors (5 μM) and stimulated with CpG-B (5 μM, 2h) as indicated were subjected to V5-immunoprecipitation. Immunoprecipitates (IP) and whole cell extracts (WCE) were analyzed by immunoblotting. (e) Immunoblots of indicated THP1::TLR9 cells. CHUK gene encodes for IKKα, MAP3K7 for TAK1 protein. (f) TNF production of indicated THP1::TLR9 cells stimulated with CpG-B (2 μM, 24h). Data show mean ± s.d. (n=3 biological replicates). (g) Immunoblots of indicated THP1::TLR9 cells stimulated with CpG-B (5 μM, 3h). (h) Schematic model representing functional homology of the SLC15A4-TASL module in mediating IRF5 activation in comparison to IRF3 adaptors STING/MAVS/TRIF. (a-g) Data are representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Figure 1. The type I interferon-inducible protein TASL is a specific interaction partner of SLC15A4.

(a) TNF production of indicated THP1 cells stimulated with R848 (5 μg/ml, 24h). Mean ± s.d. (n=3 biological replicates). (b,e) Interaction networks of (b) SLC15A4 and deletion mutants and (e) TASL identified by TAP–LC–MS/MS. Baits: red, prey proteins (SAINT FDR <1%): blue or grey if present in CRAPome database. Interactions represented as edges, line width corresponding to enrichment factor calculated by SAINT. (c) Immunoblots of THP1 cells stimulated (16h) with LPS (100 ng/ml), Pam3CSK4 (100 ng/ml), interferon β (20 ng/ml) or interferon γ (20 ng/ml). (d) Immunoblots of lysates from monocyte-derived macrophages (moM) and dendritic cells (moDC) stimulated with interferon β (20 ng/ml, 16h) treated with PNGase F as indicated. (f,g) Immunoprecipitates (IP:HA) and whole cell extracts (WCE) from (f) transduced THP1 or (g) transiently transfected HEK293T cells analyzed by immunoblotting. (h) Immunoprecipitates (IP: indicated antibodies) and WCE from indicated THP1 cells were analyzed by immunoblotting. (a,c-d,f-h) Data representative of (a) five or (c-d,f-h) two independent experiments. For gel source data, see Supplementary Figure 1.

Figure 2. TASL and SLC15A4 are required for endolysosomal TLR7/8 function.

(a) Immunoblots of THP1 cell lines. Lysates treated with PNGase F as indicated. (b) Transcriptional profiles of unstimulated and R848-treated (5 μg/ml, 6h) THP1 cell lines. Genes significantly up-regulated (FC: fold-change, DESeq2 adjusted p-value < 0.05, n=3 biological replicates) upon R848 treatment in control (sgRen) shown. (c) Gene ontology (GO) enrichment analysis (two-sided Fisher’s Exact Test, p-value adjusted for multiple testing) for R848-induced genes in control THP1 cells as defined in (b). X-axis: fold enrichment of GO terms in set of upregulated genes compared to all genes expressed (counts per million > 1). Y-axis: significance of enrichment (-log₁₀ of p-value adjusted for multiple testing). Color: fraction of R848-induced genes included in corresponding GO term. (d) Transcription factor enrichment analysis (two-sided Fisher’s Exact Test, p-value adjusted for multiple testing) of R848-induced genes in control THP1 cells as defined in (b). Background set: all expressed genes (counts per million > 1). (e,f) Cytokine production of THP1 cells stimulated (24h) with R848 (5 μg/ml) or Pam3CSK4 (100 ng/ml). Mean ± s.d. (n=3 biological replicates). (g,h) TNF production of siRNA-transfected CD14⁺ monocytes stimulated with R848 (5 μg/ml, 24h) from seven healthy donors. F: female, M: male donors. Circles represent (g) relative (normalized to siCTRL) or (h) absolute TNF levels from seven healthy donors as mean of triplicates (n=7 donors). (g) Lines indicate means over seven donors. (h) Log₂-fold changes, relative to siCTRL, of all 7 donors (n=7 donors) tested using paired, two-sided t-test. (a,e,f) Data representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Figure 3. TASL and SLC15A4 deficiency selectively impairs IRF5-dependent endolysosomal TLR signaling.

(a) Cytokine production of indicated THP1::TLR9 cells unstimulated or stimulated (24h) with CpG-A/B (5 μM) or R848 (5 μg/ml). (b) Immunoblots of indicated THP1::TLR9 cells stimulated with CpG-B (5 μM, 0-3h). (c) Fraction of CpG-B-induced genes (2 μM, 6h; DESeq2 adjusted p-value < 0.05, n=3 biological replicates) affected in IRF3, IRF5 or IRF7-deficient THP1::TLR9 cells compared to control (sgRen). (d) Transcriptional profiles of unstimulated and CpG-B-treated (2 μM, 6h) THP1::TLR9 cell lines. Genes significantly up-regulated (DESeq2 adjusted p-value < 0.05, n=3 biological replicates) by CpG-B in control (sgRen) shown. (e) Upset plot representing number of CpG-B-induced genes commonly affected by sgRNAs. 10 largest sets shown. (f) Heatmap representing 20 most induced genes by CpG-B in control THP1::TLR9 cells significantly (DESeq2 adjusted p-value < 0.05, n=3 biological replicates) affected by SLC15A4, TASL and IRF5 knockout, related to (d,e). (g) Cytokine production of indicated CAL-1 cells stimulated (24h) with R848 (5 μg/ml) or CpG-B (5 μM). (h,i) Immunoblots of (h) knockout or (i) reconstituted CAL-1 cells stimulated with R848 (5 μg/ml, 0-3h) as indicated. (a,g) Mean ± s.d. (n=3 biological replicates). (a,b,g,h,i) Data representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Figure 4. TASL is a pLxIS motif-containing adaptor required for endolysosomal TLR-induced IRF5 activation.

(a) Immunoblots of indicated THP1 cells. Bar graphs represent TNF production following R848 stimulation (5 μg/ml, 24h). (b) TASL-deficient THP1 cells reconstituted with indicated mutants were stimulated with R848 (5 μg/ml, 24h). Bar graphs show relative TNF secretion or PD-L1 expression normalized to wild type cDNA–reconstituted cells (mean ± s.d., n=2 independent experiments). Identity: fraction of evolutionary conserved amino acids in human TASL shown in Extended Data Fig. 2a. (c,d) Domain organization and multiple sequence alignment of TASL/IRF5 homology region. DBD: DNA-binding domain, IAD: IRF association domain, SR: serine-rich region. (e) Alignment of pLxIS motifs in indicated proteins. (f,h) Immunoprecipitates (IP:V5) and whole cell extracts (WCE) from indicated THP1::TLR9 cell lines stimulated with CpG-B (5 μM, 2h) analyzed by immunoblotting. sgTS: sgTASL-1. (g) Bait-normalized log₂(abundance) of indicated proteins, relative to the mean of unstimulated SLC15A4 E465K samples, determined by MS in V5-immunoprecipitates generated as in (f). Lines indicate median, n=3 biological replicates, two-sided Welch’s t-test. (i) R848-induced TNF production (5μg/ml, 24h), STAT1 and IRF5 phosphorylation (5 μg/ml, 3h) in indicated THP1 cell lines. Lysates were analyzed by immunoblot. (j) Immunoblots of reconstituted CAL-1 cells stimulated with R848 (5 μg/ml, 0-3h). (k) Schematic model of SLC15A4/TASL-dependent IRF5 activation by TLR7-9. (a,i) Bar graphs show mean ± s.d. (n=3 biological replicates). (a,f,h-j) Data representative of two independent experiments. For gel source data, see Supplementary Figure 1.