Quantitative proteomics reveals protein dysregulation during T cell activation in multiple sclerosis patients compared to healthy controls

Background Multiple sclerosis (MS) is an autoimmune, neurodegenerative disorder with a strong genetic component that acts in a complex interaction with environmental factors for disease development. CD4+ T cells are pivotal players in MS pathogenesis, where peripherally activated T cells migrate to the central nervous system leading to demyelination and axonal degeneration. Through a proteomic approach, we aim at identifying dysregulated pathways in activated T cells from MS patients as compared to healthy controls. Methods CD4+ T cells were purified from peripheral blood from MS patients and healthy controls by magnetic separation. Cells were left unstimulated or stimulated in vitro through the TCR and costimulatory CD28 receptor for 24 h prior to sampling. Electrospray liquid chromatography-tandem mass spectrometry was used to measure protein abundances. Results Upon T cell activation the abundance of 1801 proteins was changed. Among these proteins, we observed an enrichment of proteins expressed by MS-susceptibility genes. When comparing protein abundances in T cell samples from healthy controls and MS patients, 18 and 33 proteins were differentially expressed in unstimulated and stimulated CD4+ T cells, respectively. Moreover, 353 and 304 proteins were identified as proteins exclusively induced upon T cell activation in healthy controls and MS patients, respectively and dysregulation of the Nur77 pathway was observed only in samples from MS patients. Conclusions Our study highlights the importance of CD4+ T cell activation for MS, as proteins that change in abundance upon T cell activation are enriched for proteins encoded by MS susceptibility genes. The results provide evidence for proteomic disturbances in T cell activation in MS, and pinpoint to dysregulation of the Nur77 pathway, a biological pathway known to limit aberrant effector T cell responses

Multiple Sclerosis Risk Allele in <i>CLEC16A</i> Acts as an Expression Quantitative Trait Locus for <i>CLEC16A</i> and <i>SOCS1</i> in CD4+ T Cells

<div><p>For multiple sclerosis, genome wide association studies and follow up studies have identified susceptibility single nucleotide polymorphisms located in or near <i>CLEC16A</i> at chromosome 16p13.13, encompassing among others <i>CIITA</i>, <i>DEXI</i> and <i>SOCS1</i> in addition to <i>CLEC16A</i>. These genetic variants are located in intronic or intergenic regions and display strong linkage disequilibrium with each other, complicating the understanding of their functional contribution and the identification of the direct causal variant(s). Previous studies have shown that multiple sclerosis-associated risk variants in <i>CLEC16A</i> act as expression quantitative trait loci for <i>CLEC16A</i> itself in human pancreatic β-cells, for <i>DEXI</i> and <i>SOCS1</i> in thymic tissue samples, and for <i>DEXI</i> in monocytes and lymphoblastoid cell lines. Since T cells are major players in multiple sclerosis pathogenesis, we have performed expression analyses of the <i>CIITA-DEXI-CLEC16A-SOCS1</i> gene cluster in CD4+ and CD8+ T cells isolated from multiple sclerosis patients and healthy controls. We observed a higher expression of <i>SOCS1</i> and <i>CLEC16A</i> in CD4+ T cells in samples homozygous for the risk allele of <i>CLEC16A</i> rs12927355. Pair-wise linear regression analysis revealed high correlation in gene expression in peripheral T cells of <i>CIITA</i>, <i>DEXI</i>, <i>CLEC16A</i> and <i>SOCS1</i>. Our data imply a possible regulatory role for the multiple sclerosis-associated rs12927355 in <i>CLEC16A</i>.</p></div

The genotype of rs12927355 associates with increased expression of <i>CLEC16A</i> and <i>SOCS1</i> in CD4+T cells.

<p>The plots show gene expression of <i>CIITA</i>, <i>DEXI</i>, <i>CLEC16A</i> and <i>SOCS1</i> relative to <i>TBP</i> in CD4+ T cells (n = 50) from MS patients (n = 27) and HCs (n = 23). The samples were sorted according to <i>CLEC16A</i> genotype of two MS-associated SNPs (A) rs12927355 (risk allele = G): GG: n = 35, AG: n = 14 and AA: n = 1, and (B) rs4780346 (risk allele = A): AA: n = 4 and AG: n = 19, GG: n = 27. Mann-Whitney U-test was performed to compare the groups. Significant <i>P</i>-values are shown in the figure. The median value in each group is indicated as a horizontal line.</p

No difference in 16p13.13 T cell expression between MS patients and healthy controls.

<p>The plots show gene expression of <i>CIITA</i>, <i>DEXI</i>, <i>CLEC16A</i> and <i>SOCS1</i> relative to <i>TBP</i> in (A) CD4+ T cells (MS: n = 28; HC: n = 26) and (B) CD8+ T cells (MS n = 17; HC: n = 23). Mann-Whitney U-test was performed to compare the groups. The median value in each group is indicated as a horizontal line.</p

Characteristics of MS patients and controls.

<p>¹ At inclusion in this study.</p><p>Abbreviations: EDSS = expanded disability status scale, S.D. = standard deviation, N/A = not applicable.</p><p>Characteristics of MS patients and controls.</p

Increased DNA methylation of SLFN12 in CD4+ and CD8+ T cells from multiple sclerosis patients.

DNA methylation is an epigenetic mark that is influenced by environmental factors and is associated with changes to gene expression and phenotypes. It may link environmental exposures to disease etiology or indicate important gene pathways involved in disease pathogenesis. We identified genomic regions that are differentially methylated in T cells of patients with relapsing remitting multiple sclerosis (MS) compared to healthy controls. DNA methylation was assessed at 450,000 genomic sites in CD4+ and CD8+ T cells purified from peripheral blood of 94 women with MS and 94 healthy women, and differentially methylated regions were identified using bumphunter. Differential DNA methylation was observed near four loci: MOG/ZFP57, HLA-DRB1, NINJ2/LOC100049716, and SLFN12. Increased methylation of the first exon of the SLFN12 gene was observed in both T cell subtypes and remained present after restricting analyses to samples from patients who had never been on treatment or had been off treatment for more than 2.5 years. Genes near the regions of differential methylation in T cells were assessed for differential expression in whole blood samples from a separate population of 1,329 women with MS and 97 healthy women. Gene expression of HLA-DRB1, NINJ2, and SLFN12 was observed to be decreased in whole blood in MS patients compared to controls. We conclude that T cells from MS patients display regions of differential DNA methylation compared to controls, and corresponding gene expression differences are observed in whole blood. Two of the genes that showed both methylation and expression differences, NINJ2 and SLFN12, have not previously been implicated in MS. SLFN12 is a particularly compelling target of further research, as this gene is known to be down-regulated during T cell activation and up-regulated by type I interferons (IFNs), which are used to treat MS