A purine metabolic checkpoint that prevents autoimmunity and autoinflammation

Abstract

Still's disease, the paradigm of autoinflammation-cum-autoimmunity, predisposes for a cytokine storm with excessive T lymphocyte activation upon viral infection. Loss of function of the purine nucleoside enzyme FAMIN is the sole known cause for monogenic Still's disease. Here we discovered that a FAMIN-enabled purine metabolon in dendritic cells (DCs) restrains CD4⁺ and CD8⁺ T cell priming. DCs with absent FAMIN activity prime for enhanced antigen-specific cytotoxicity, IFNγ secretion, and T cell expansion, resulting in excessive influenza A virus-specific responses. Enhanced priming is already manifest with hypomorphic FAMIN-I254V, for which ∼6% of mankind is homozygous. FAMIN controls membrane trafficking and restrains antigen presentation in an NADH/NAD⁺-dependent manner by balancing flux through adenine-guanine nucleotide interconversion cycles. FAMIN additionally converts hypoxanthine into inosine, which DCs release to dampen T cell activation. Compromised FAMIN consequently enhances immunosurveillance of syngeneic tumors. FAMIN is a biochemical checkpoint that protects against excessive antiviral T cell responses, autoimmunity, and autoinflammation.

Keywords: NADH/NAD(+) reductive stress; T cell priming; autoimmunity; dendritic cells; membrane trafficking; purine nucleotide cycle.

Graphical abstract

Figure 1

FAMIN deficiency augments T cell responses to influenza A virus (A) Percentage weight loss of Famin^p.254I, Famin^p.254V, Famin^p.284R mice, which are engineered to express fully active, hypomorphic, and inactive FAMIN, respectively, following infection with influenza A virus (IAV) H3N2, strain A/X-31 (n = 6/11/6). (B) Average number of TUNEL⁺ cells in lungs of Famin^p.254I, Famin^p.254V, and Famin^p.284R mice 7 days post-infection (n = 6/9/6). (C) Plasma IFNγ and IL-10 levels 7 days after IAV infection (n = 6/11/6). Note IL-12p70 was below the detection limit. (D and E) Absolute numbers of IAV NP^366-374 tetramer⁺ CD8⁺ T cells in Famin^p.254I, Famin^p.254V, and Famin^p.284R (D), or CD3⁺CD8⁺NP^366-374 and PA^224-233 tetramer⁺ cells in Famin^WT and Famin^ΔDC (E) bronchoalveolar lavage fluid (BAL) 7 days after infection (n = 6/11/6; 15/14). (F) IAV M protein gene expression in lung tissue of Famin^p.254I, Famin^p.254V, and Famin^p.284R mice 7 days after infection (n = 6/11/6). Data represented as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 (repeated-measures one-way ANOVA, one-way ANOVA, or unpaired two-tailed Student’s t test where appropriate). See also Figure S1.

Figure 2

DC FAMIN restrains CD4⁺ and CD8⁺ T cell responses (A–C) IFNγ release and OT-I T cell proliferation indices after 72 h of co-culture with splenic Famin^WT or Famin^ΔDC CD11c⁺ DCs pulsed with OVA^257-264 peptide (A), ovalbumin (B), or UV-irradiated bm1 T OVA mouse embryonic fibroblasts (C) (n = 3). (D and E) Specific cytotoxicity against OVA^257-264-pulsed wild-type splenocytes (D), and IFNγ and granzyme B release (E) of OT-I T cells that had been primed with Famin^WT or Famin^ΔDC splenic DCs pulsed with OVA^257-264 (n = 3). (F) IFNγ secretion from re-stimulated (OVA^257-264 for 5 h) OT-I T cells after 72 h of priming with Famin^p.254I, Famin^p.254V, Famin^p.284R, or Famin^−/− BM-derived cDC1 pulsed with OVA^257-264 (n = 3). (G) IFNγ secretion after 5 h anti-CD3/CD28 re-stimulation of OT-I T cells that had been differentiated into T_E and T_EM cells, following 72 h of priming by Famin^p.254I, Famin^p.254V, Famin^p.284R, and Famin^−/− BM-derived cDC1 pulsed with OVA^257-264 (n = 3). (H) OVA^257-264-specific cytotoxicity, granzyme B, and IFNγ secretion of splenocytes of Famin^ΔDC and Famin^WT mice that had been adoptively transferred with naive OT-I T cells and immunized with ovalbumin 72 h earlier (n = 3). (I) Proliferation indices of OT-II T cells 96 h after priming with OVA^323-339-pulsed splenic Famin^+/+ and Famin^−/− DCs (n = 3). (J and K) IFNγ (J) and IL-2 (K) in supernatants of OT-II cells 96 h after priming with OVA^323-339-pulsed splenic Famin^+/+ and Famin^−/− DCs (n = 3). (L) Percentage IFNγ⁺ OT-II T cells after restimulation with anti-CD3/CD28, following priming with OVA^323-339-pulsed BM-derived cDC2 7 days earlier (n = 3). (M) Proliferation indices of OT-II T cells adoptively transferred into Famin^ΔDC and Famin^WT mice 72 h after immunization with ovalbumin (n = 3). Data represented as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 (one-way ANOVA or unpaired two-tailed Student’s t test where appropriate). See also Figure S2.

Figure 3

FAMIN controls DC metabolism and tunes antigen uptake and presentation without a transcriptional signature (A) Differentially expressed genes between Famin^−/− and Famin^+/+ BM-derived cDC1s (n = 4; GEO: GSE126473). (B) Heatmap of purine nucleotide levels in Famin^p.254I, Famin^p.254V, and Famin^p.284R cDC1s (n = 6/5/6); for details, see Table S3. (C) Differential metabolite abundance between Famin^p.254I and Famin^p.284R BM-derived cDC1s; identifiable differential LC-MS features (positive and negative ionization modes) highlighted in red and annotated (n = 5). (D) Percentage AF647-OVA⁺Famin^p.254I, Famin^p.254V, or Famin^p.284R splenic CD11c⁺ DCs following uptake of AF647-OVA for indicated times (n = 3). (E) Endosome-to-cytosol transfer in β-lactamase-loaded Famin^p.254I and Famin^p.284R cDC1s (n = 6/6, from 3 mice per genotype). (F) Percentage splenic CD11c⁺ DCs staining positive for OVA^257-264 bound to H-2K^b, after incubation with OVA^257-264 for indicated times (n = 3). (G) Schematic depiction of the IMP–S-AMP–AMP cycle and IMP–XMP–GMP cycle, with relationship to FAMIN products and substrates. ADSS, adenylosuccinate synthase; ADSL, adenylosuccinate lyase; AMPD, AMP deaminase; IMPDH, IMP dehydrogenase; GMPS, GMP synthase; GMPR, GMP reductase; HPRT, hypoxanthine guanine phosphoribosyltransferase; APRT, adenine phosphoribosyltransferase, ADA, adenosine deaminase; PNP, purine nucleoside phosphorylase; MTA, methylthioadenosine; MTAP, MTA phosphorylase; SAM, S-adenosylmethionine; dcSAM, decarboxylated SAM. (H) Oxygen consumption rate (OCR) of Famin^p.254I, Famin^p.254V, and Famin^p.284R BM-derived cDC1s. Basal OCR followed by oligomycin A (Oligo), FCCP, and rotenone plus antimycin A (Rot + Ant) (n = 16–18, from 3 mice per genotype). (I) Basal extracellular acidification rate (ECAR) of Famin^p.254I, Famin^p.254V, and Famin^p.284R BM-derived cDC1s (n = 16–18, from 3 mice per genotype). (J) Secreted lactate levels in supernatants of Famin^p.254I, Famin^p.254V, and Famin^p.284R splenic DCs after 3 h incubation in OptiMEM medium (n = 8–9, from 3 mice per genotype). (K) Cytoplasmic pH (pH_c) of Famin^p.254I, Famin^p.254V, and Famin^p.284R splenic DCs measured using pHrodo indicator probe (n = 12, from 3 mice per genotype). (L) Fractional labeling of cellular aspartate as [¹³C₄¹⁵N₁] isotopomer in Famin^p.254I, Famin^p.254V, and Famin^p.284R BM-derived cDC1 following a 3 h pulse with [¹³C₄ ¹⁵N₁] aspartate (n = 6, from 3 mice per genotype). (M) Fractional labeling of [¹³C₄] malate and levels of unlabeled (M+0) malate following a 3 h pulse of Famin^p.254I, Famin^p.254V, and Famin^p.284R cDC1s with [¹³C₄] malate (n = 9/9/5, from 3/3/2 mice per genotype). (N) Total levels of aspartate, IMP, succinyl-AMP, and AMP following a 3 h pulse of Famin^p.254I, Famin^p.254V, and Famin^p.284R cDC1s with [¹³C₄] malate (n = 9/9/5, from 3/3/2 mice per genotype). Data represented as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 (one-way ANOVA or unpaired two-tailed Student’s t test where appropriate). See also Figure S3.

Figure 4

Adenine-guanine nucleotide interconversion paces antigen uptake and T cell priming (A–D) Percentage AF647-OVA⁺ cDC1s (A, B, and D) or splenic DCs (C) following incubation with AF647-OVA for 30 min in the presence of L-alanosine (A), hadacidin (B), 6-mercaptopurine (6-MP) (C), or Cpd3 (D) (n = 3–6, 3 mice per genotype; none of the treatments affected cell viability; please note control panels are shared between B and D). (E and F) Proliferation indices, CFSE overlays (E), and IFNγ released (F) from OT-I T cells co-cultured for 72 h with Famin^p.254I splenic DCs pulsed with ovalbumin 48 h after nucleofection with ctrl or Adsl, Adss, and Ampd2/Ampd3 siRNAs (n = 6, 3 mice per siRNA). (G) Heatmap of purine nucleotide levels in Famin^p.254I cDC1s, treated with Cpd3 or vehicle for 18 h (n = 5); normalized peak integrals and fold changes are shown in Table S5. (H and I) Percentage AF647-OVA⁺ splenic DCs of indicated genotypes after incubation with mycophenolic acid (MPA) (H) or psicofuranine (I) (n = 18, from 3 independent experiments in H; n = 6, 3 mice per genotype in I). (J) Fractional incorporation into indicated GMP isotopomers following a 3 h pulse of Famin^p.254I, Famin^p.254V, and Famin^p.284R cDC1s with [¹⁵N₂¹³C₅] glutamine (n = 7/6/6, 3 mice per genotype). (K–M) IFNγ released from OT-I T cells co-cultured for 72 h with ovalbumin-pulsed Famin^p.254I (K), Famin^p.254V (L), or Famin^p.284R (M) splenic DCs 48 h after nucleofection with Gmpr2, Gmps, Impdh1/Impdh2, or ctrl siRNAs (n = 6, 3 mice per group). (N–P) Proliferation indices from OT-I T cells co-cultured for 72 h with Famin^p.254I (N), Famin^p.254V (O), and Famin^p.284R (P) splenic DCs pulsed with ovalbumin 48 h after nucleofection with ctrl or Gmpr2, Gmps, Impdh1, and Impdh2 siRNA (n = 6, 3 mice per siRNA). Data represented as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 (one-way ANOVA or unpaired two-tailed Student’s t test where appropriate). See also Figure S4.

Figure 5

IMPDH-dependent NADH/NAD⁺ redox state controls the pace of antigen uptake and MHC I recycling (A) Percentage AF647-OVA⁺ splenic DCs of indicated genotypes following incubation with guanine (n = 6, 3 mice per genotype). (B) pH_c of Famin^p.254I, Famin^p.254V, and Famin^p.284R cDC1s in the presence of MPA (n = 8–12, 3 mice per genotype). (C) pH_c of Famin^p.254I, Famin^p.254V, and Famin^p.284R splenic DCs in the presence of L-alanosine (n = 9, 3 mice per genotype). (D) OCR of Famin^p.254I and Famin^p.284R BM-derived cDC1s in the presence of MPA or vehicle control. Basal OCR and OCR following addition of oligomycin A (Oligo), FCCP, and Rot + Ant (n = 7–14, 3 mice per genotype). (E) Basal ECAR of Famin^p.254I and Famin^p.284R BM-derived cDC1in the presence of MPA or vehicle control (n = 16–18, 3 mice per genotype). (F) Ratio of secreted lactate to pyruvate, reflective of free cytosolic NADH/NAD⁺, in supernatants of Famin^p.254I and Famin^p.284R splenic DCs matured overnight in RPMI-1640/10% FBS (n = 9, from 3 mice per genotype). (G) Schematic depicting the lactate dehydrogenase (LDH) reaction, in which pyruvate is converted to lactate with regeneration of NAD⁺; α-ketobutyrate acts as an alternative electron acceptor and is converted to α-hydroxybutyrate. (H) Percentage AF647-OVA⁺ splenic DCs of indicated genotypes following incubation with pyruvate or α-ketobutyrate (AKB) overnight and replenished for the time of the assay (n = 6, 3 mice per genotype). (I) Percentage AF647-OVA⁺Famin^p.254I splenic DCs following overnight incubation with pyruvate or AKB alone or in presence of Cpd3 (n = 6, 3 mice per genotype). (J) Percentage of MHC I recycled in Famin^p.254I and Famin^p.284R BM-derived cDC1s over time (n = 6, 3 mice per genotype). (K) Percentage of MHC I recycled in Famin^p.284R BM-derived cDC1s at indicated times following overnight incubation with AKB or control (n = 3–5, 3 mice per genotype). Data represented as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 (one-way ANOVA or unpaired two-tailed Student’s t test where appropriate). See also Figure S5.

Figure 6

The FAMIN catalytic product inosine released from DCs dampens T cell activation during priming (A) IFNγ secretion from naive OT-I T cells activated by anti-CD3/CD28 in presence of 24 h supernatants of Famin^−/− or Famin^+/+ CD11c⁺ splenic DCs (n = 3). (B and C) Gene set enrichment analysis (GSEA) of RNA-seq dataset of naive OT-I T cells activated by anti-CD3/CD28 for 24 h in the presence of Famin^−/− or Famin^+/+ splenic DC supernatant; data depict enrichment of the Hallmark gene sets “oxidative phosphorylation” (B) and “Myc Targets V2” (C). (D) IFNγ secretion from OT-I T cells activated by anti-CD3/CD28 for 72 h in the presence of <3 kDa cut-off filtrates of supernatants from Famin^+/+ or Famin^−/− splenic DCs cultured for 3 h in RPMI-1640/10% FBS (n = 3). (E) IFNγ secretion, proliferation indices, and CFSE overlays from OT-I T cells activated with anti-CD3/CD28 for 72 h in the presence of supernatants from Famin^p.254I, Famin^p.254V, and Famin^p.284R splenic DCs cultured for 3 h in OptiMEM (n = 3). (F) IFNγ from 72 h anti-CD3/CD28 stimulated naive OT-II T cells cultured in the presence of 3 h supernatant from Famin^p.254I, Famin^p.254V, and Famin^p.284R splenic DCs (n = 6, 3 mice per genotype). (G) Differential abundance of LC-MS features in supernatants from Famin^−/− versus Famin^+/+ splenic DCs cultured for 3 h in OptiMEM (n = 8–9, 3 mice per genotype). Data depicted as volcano plot showing p value and log₂ fold change for each detected LC-MS feature. (H) Representative extracted chromatograms, using normalized peak intensity, showing inosine detection in supernatants from Famin^−/− and Famin^+/+ splenic DCs and corresponding standard. (I) Differential LC-MS features in supernatants from Famin^p.254I and Famin^p.284R splenic DCs cultured for 3 h in OptiMEM (n = 8–9, 3 mice per genotype). Data depicted as volcano plot showing p value and log₂ fold change for each detected LC-MS feature; FDR-adjusted p value is depicted. (J and K) Relative inosine levels released in supernatants from Famin^−/− or Famin^+/+ (J) or Famin^p.254I, Famin^p.254V, and Famin^p.284R (K) splenic DCs, cultured for 3 h in OptiMEM (n = 8–9, 3 mice per genotype). (L and M) IFNγ secretion (L) and proliferation indices (M) of naive OT-I T cells stimulated with anti-CD3/CD28 in the presence of indicated concentrations of inosine for 72 h (n = 3). (N and O) Division index (N) and IFNγ secretion (O) from Famin^p.254I and Famin^p.284R splenic DCs pulsed with ovalbumin and co-cultured with OT-I T cells in presence of indicated spiked-in concentrations of inosine into RPMI-1640 (concentration from 0.1 to 5 nM, with 5 nM reflecting the lower end of differences in inosine release between Famin^p.254I and Famin^p.284R DC supernatants, such as in Figure S6H) (n = 3–6, from 3 mice per genotype). (P and Q) IFNγ secretion (P) and proliferation indices (Q) of OT-I T cells activated by anti-CD3/CD28 for 72 h in the presence of CGS21680 or SCH58261 and supernatants from Famin^p.254I, Famin^p.254V, and Famin^p.284R splenic DCs cultured for 3 h in OptiMEM (n = 6, 3 mice per genotype). Data represented as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 (one-way ANOVA or unpaired two-tailed Student’s t test where appropriate). See also Figure S6.

Figure 7

FAMIN-dependent conversion of extracellular hypoxanthine into inosine (A) FAMIN-catalyzed enzymatic conversion of [¹³C₅¹⁵N₄] hypoxanthine to [¹³C₅¹⁵N₄] inosine. (B) Cellular [¹³C₅¹⁵N₄] inosine in Famin^p.254I, Famin^p.254V, and Famin^p.284R splenic DCs pre-equilibrated in OptiMEM for 3 h before a 3 h pulse with [¹³C₅¹⁵N₄] hypoxanthine in OptiMEM (n = 6, 3 mice per genotype). (C) [¹³C₅¹⁵N₄] inosine, and its fractional incorporation, in supernatants of Famin^p.254I, Famin^p.254V, and Famin^p.284R splenic DCs pre-equilibrated in OptiMEM for 3 h before a 3 h pulse with [¹³C₅¹⁵N₄] hypoxanthine in OptiMEM (n = 18, 3 mice per genotype). (D) Unlabeled (M+0) inosine in supernatants of Famin^p.254I, Famin^p.254V, and Famin^p.284R splenic DCs following a 3 h pulse with [¹³C₅¹⁵N₄] hypoxanthine in OptiMEM (n = 18, 3 mice per genotype). (E) Representative extracted chromatograms, showing peak corresponding to inosine for indicated standards and for RPMI-1640/10% FBS medium, in which inosine was below the quantification limit. (F–H) Inosine levels released into supernatants from Famin^p.254I, Famin^p.254V, and Famin^p.284R splenic DCs, pre-treated with MPA (F), hadacidin (G), or Cpd3 (H) for 18 h and cultured for 3 h in OptiMEM with inhibitors replenished in medium (n = 9, 3 mice per genotype). (I) Tumor volume over time, and on day 26, after subcutaneous inoculation of Famin^p.254I, Famin^p.254V, and Famin^p.284R mice with 2.5 × 10⁴ LL2-OVA cells (n = 6/7/7, three mice were sacrificed due to fight wounds on day 23 during the blinded phase of the experiment; genotype was post hoc identified as Famin^p.284R). (J) Tumor diameter over time and on day 30 post inoculation of Famin^p.254V and Famin^p.284R mice subcutaneously injected with 2 × 10⁴ LL2-OVA cells (n = 10/8). Data represented as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 (one-way ANOVA or unpaired two-tailed Student’s t test where appropriate). See also Figure S7.