Protein dysregulation in immune cells of multiple sclerosis patients

Multiple sclerosis (MS) is a complex, autoimmune and inflammatory disease affecting the central nervous system. The pathogenesis of MS is not completely understood, but T cell activation is believed to be an important part of disease etiology. MS susceptibility is provided through a combination of several single contributions involving environmental exposures and multiple genetic risk variants. To date, more than 200 MS associated risk variants have been identified, many of which are linked to immune cell reactions in T cells. In the current project, we aimed at (i) using available proteomic data from CD4+ and CD8+ T cells of genome-wide genotyped MS patients and healthy controls to identify novel MS- linked proteins or pathways. Secondly, (ii) we aimed at collecting new samples from activated CD4+ T cells from the same groups for proteomics profiling by mass spectrometry. (i) A proteomic data set from CD4+ and CD8+ T cells was utilized to correlate the genotype at MS susceptibility variants with the expression of proteins encoded by genes located 100kb upstream and downstream of the MS risk variants. Furthermore, pathway analysis was performed on proteins that were differentially expressed between MS patients and healthy controls in both cell types. (ii) Live CD4+ T cells are stored on liquid nitrogen, and cells from 28 female relapsing-remitting MS patients and 28 healthy individuals were thawed and activated in vitro with -CD3 and -CD28 antibodies. The cells were analyzed by flow cytometry for cell surface expression of the T cell activation marker CD69 and with a LIVE/DEAD stain distinguishing live from dead cells. Cells with >50 % CD69 positive cells after activation and with >70 % viability were sent for liquid chromatography-tandem mass spectrometry analysis to analyze the proteome of the samples. (i) Two novel protein quantitative trait loci (pQTL) candidates were identified, i.e. rs1800693 - CD9 and rs137955 - UQCRFS1P1. 26 proteins were differentially expressed in both CD4+ and CD8+ T cells between the groups, and pathway analysis did not identify any specific enriched biological pathways. The identified pQTL candidates might have relevance for MS disease, and can inspire for functional studies to seek a broader understanding of the effect of genetic risk variants, as well as the mechanisms behind MS development. (ii) A total of 20 samples from MS patients and 20 samples from healthy controls fulfilled the inclusion criteria, and were included in the proteomic analysis. Thus, samples from un-activated and cells activated for 24 hours were shipped to the proteomics core facility in Bergen. The samples are lysed an prepared for proteomic profiling, but the results of the analysis is not available yet, due to the Covid-19 situation. Whether the proteomic analysis of activated CD4+ T cells lead to identification of proteins and pathways of importance for MS, remains to be shown

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

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