Fig 1.
The IBD-protective rs6478109:A allele is associated with increased TNFSF15 expression.
(A) Regional association plots for IBD (European ancestry cohort from Liu et al [4]) and TNFSF15 expression (n = 39 healthy individuals and 80 IBD patients). (B) TNFSF15 mRNA expression was measured in ex vivo monocytes relative to B2M by qPCR in a separate cohort of healthy individuals and plotted versus rs6478109 genotype (GG, n = 10; GA, n = 16; AA, n = 9). Expression is reported as ΔCt, each point represents an individual, lines represent mean values, and p value is from linear regression.
Fig 2.
Monocyte but not T cell TNFSF15 expression is associated with genotype after stimulation.
(A) TNFSF15 mRNA expression was measured relative to B2M in monocytes stimulated for 4 hours with immune complex (rs6478109 genotype: GG, n = 6; GA, n = 10; AA, n = 5), intracellular poly(I:C) (GG, n = 8; GA, n = 10; AA, n = 8), or LPS (GG, n = 8; GA, n = 11; AA, n = 7). Expression is reported as ΔCt, each point represents an individual, lines represent mean values, and p values are from linear regression. (B) As (A) for CD4+ (GG, n = 10; GA, n = 16; AA, n = 9) and CD8+ (GG, n = 9; GA, n = 10; AA, n = 9) T cells stimulated for 24 hours. (C) Allelic ratios in cDNA from stimulated cells from heterozygous individuals are compared with those in genomic DNA from the same individuals: monocytes stimulated with immune complex (n = 7), intracellular poly(I:C) (n = 10) and LPS (n = 12), and (D) anti-CD3/anti-CD28-stimulated CD4+ (n = 11) and CD8+ (n = 10, genomic DNA as for panel (C) intracellular poly(I:C)) T cells. Ratios for immune complex- and LPS-stimulated monocytes and stimulated CD4+ T cells were measured using tag SNP rs4246905 (ratio of T/C reported), while ratios for poly(I:C)-stimulated monocytes and stimulated CD8+ T cells were measured using tag SNP rs4263839 (ratio of A/G reported). All individuals were heterozygous at both rs6478109 and the tag SNP. rs4263839:A and rs4246905:T are in phase with the IBD-protective allele rs6478109:A in 1000 Genomes EUR subjects. Lines represent mean values and p values are from Welch’s t-test.
Fig 3.
Genotype is associated with de novo TNFSF15 protein production in stimulated monocytes.
(A) An example of gating for TNFSF15+ monocytes after 4 hours immune complex stimulation is depicted (left). Black line = monoclonal anti-TNFSF15; grey shading = isotype control. Percentages of TNFSF15+ monocytes after immune complex stimulation are quantified for GG (n = 12) and AA (n = 12) homozygous individuals (right). (B) Soluble TNFSF15 was measured in supernatants of monocytes from GG (n = 12) and AA (n = 12) homozygous individuals after the indicated 4-hour stimulations by custom anti-TNFSF15 Bio-Plex assay. p values are from Mann-Whitney test. (C) Monocytes were stimulated with immune complex for four hours in the presence of actinomycin D (ActD) or dimethyl sulfoxide (DMSO) control. ND indicates none detected. (D) As (C) in the presence or absence of cycloheximide (CHX). (C) and (D) are representative of two independent experiments each.
Fig 4.
The eQTL causal SNP resides upstream of TNFSF15.
(A) The TNFSF15 locus on chromosome 9 is depicted, marking SNPs used for functional fine-mapping. (B) Immune complex-stimulated monocytes from individuals heterozygous at rs6478109 and rs4263839 but homozygous at rs4246905 (n = 4) were examined for allele-specific expression. The box on the transcription arrow indicates the SNP used for measuring allelic imbalance. (C) As (B) with individuals homozygous at rs6478109 and rs4263839 but heterozygous at rs4246905 (n = 4). (D) As (B) with individuals homozygous at rs6478109 but heterozygous at rs4263839 and rs4246905 (n = 3). Lines represent mean values and p values are from Welch’s t-test.
Fig 5.
Genetically regulated TNFSF15 controls costimulatory capacity.
(A) CD8-depleted PBMC were stained with proliferation dye eFluor670 and stimulated with 1 μg/mL anti-CD3 with and without 10 μg/mL anti-TNFSF15 for 48 hours. Flow cytometry plots depict cells gated on live, CD4+ cells. (B) CD25+ cells were measured as in (A) in samples from rs6478109 genotyped individuals (GG, n = 8; AA, n = 7). (C) CD25+ divided cells were measured in the same samples as in (B). p values from Mann-Whitney test.
Fig 6.
Monocyte-derived macrophages also favor expression of the IBD-protective allele.
(A) Peripheral blood monocytes from rs6478109 heterozygotes were differentiated into macrophages in the presence of M-CSF (n = 10) or (B) GM-CSF (n = 7) before examining allele-specific expression. (C) Macrophages derived in (A) were stimulated with LPS for 4 hours before examining allele-specific expression (n = 10, genomic DNA as for panel (A)). ASE was measured at rs4263839 (ratio of A/G reported) in the first intron of TNFSF15. All individuals were heterozygous at both rs4263839 and rs6478109. rs4263839:A is in phase with the IBD-protective allele rs6478109:A in 1000 Genomes EUR subjects.