Author Of 1 Presentation
YI01.01 - Single-cell RNAseq of multiple sclerosis cerebrospinal fluid identifies T cell and myeloid subsets that are over-represented at disease onset
Abstract
Background
Immune cells play an important role in the pathogenesis of MS. However, our knowledge of the diversity of immune cell types and states in health and disease has been limited by the restrictions of traditional oligo-marker immunological approaches. Single-cell RNA sequencing (scRNA-seq) provides new means for discovery and characterization of immune cells by measuring expression levels of thousands of genes simultaneously at the single cell level.
Objectives
Here, we investigated the immune cell composition of cerebrospinal fluid (CSF), as the most accessible immune compartment in contact with the central nervous system, using scRNA-seq in MS and non-MS neuroinflammatory conditions (ONID).
Methods
Fresh CSF samples were collected at the time of diagnostic lumbar puncture from 8 untreated RRMS and 7 ONID patients. In addition, to examine the context of the reconstituted CSF after ocrelizumab treatment, we sampled 6 progressive MS patients on ocrelizumab for >1 year, 5 months after their last dose. A replication set of 3 untreated RRMS and 3 ONID are also collected.
Results
After removing doublets, RBCs, and low-quality cells, 59,288 cells were remained for analysis. Data were integrated using canonical correlation analysis. Over-clustering using ‘Seurat’ and iterative merging of clusters with more similar gene expression using ‘SCCAF’ resulted in 13 major clusters: 1 αβ-T cell cluster (consisting of 24 subclusters), 4 other T (NK, NKT, γδ-T), 5 myeloid (microglia-like monocytic, myeloid DC, granulocytes), B cells, plasmablasts and plasmacytoid DC.
Consistent with earlier smaller studies, we observed subsets of myeloid cells with microglia-like features (3% frequency). Using our reference brain-derived microglia scRNA-seq dataset (221,126 cells), we have characterized the microglial subtypes to which the CSF cells most resemble. Interestingly, these are part of the 4 myeloid subtypes which percentage were reduced in newly diagnosed RRMS relative to ONID. In addition, 3 CD4+ and 1 CD8+ memory T cell subsets were increased in RRMS. If replicated, these intriguing differences could guide our understanding of earliest stages of MS vs. other elements of the differential.
B cell component was largely reconstituted in the ocrelizumab group, and the patients had a CD4+ and CD8+ T cell pattern largely similar to ONID (vs RRMS). However, myeloid cell percentages were increased even more in this group. Further studies are needed to resolve whether these changes are due to progression vs. age vs. ocrelizumab.
Conclusions
Our data offer a new level of resolution in cell population shifts and suggest that measuring certain combinations of subsets could be useful clinically. In addition to these results, we will present on gene-level expression differences and a first draft of a CSF molecular network map across the 3 conditions.
Author Of 1 Presentation
LB1250 - Neuronal integrity is associated to peripheral leukocyte dysfunction in asymptomatic Multiple Sclerosis (MS) first-degree relatives (ID 2136)
Abstract
Background
Genetic studies suggest that peripheral immune dysfunctions occurs first in the causal chain of events leading to MS. However, these changes are typically not appreciated when patients present with their first symptom or with an asymptomatic inflammatory lesion; we have only sparsely characterized individuals before they develop disease. Thus, the earliest molecular events leading to damage of the CNS parenchyma remain unknown.
Objectives
To investigate the correlation between a marker of neuronal integrity (Neurofilament light chain, NfL) and the transcriptional profile of peripheral immune cells during the pre-symptomatic phase in a cohort of first-degree family members of MS patients.
Methods
Blood samples were collected from 124 asymptomatic first-degree relatives of MS patients part of the Genes & Environment in MS (GEMS) study, 7 MS patients, and 11 patients with other neuroimmune diseases (ONID), age (20-54). Serum samples were used to assess NfL levels using the SIMOA platform. Cryopreserved peripheral blood mononuclear cells (PBMC) were used to generate RNA-sequencing data. ‘CIBERSORT’ was used to estimate the proportions of major PBMC cell types (CD4 and CD8 T, monocyte, B, and NK) from the gene expression data. ‘WGCNA’ was used to to identify PBMC co-expressed gene modules. Available genotyping data were used to calculate a polygenic MS risk score for each individual.
Results
Serum NfL levels from GEMS individuals increased with age (p=2E-14), and were significantly lower than MS and ONID patients, adjusting for the effects of age and sex. NfL was not elevated in high-risk (high polygenic score) vs. low-risk family member subgroups.
After batch variation and technical variable correction 114 GEMS subjects qualified for PBMC bulk RNA-sequencing analysis. ‘WGCNA’ identified 37 co-expressed gene modules, 4 of which were associated with NfL levels (FDR-adjusted p<0.05), adjusting for effects of age, sex and estimated cell type proportions. One particular module is enriched in myeloid cells, and myeloid-mediated immunity and myeloid activation pathways; an association that persisted after accounting for the proportion of different cell types. The other 3 modules were all enriched for genes upregulated in chronic myelogenous leukemia and downregulated gene targets of KLF1 transcription factor.
Conclusions
The transcriptional programs of some peripheral immune cells strongly correlate with NfL levels; ongoing data replication. These results implicate a cross-talk between the peripheral immune system and the CNS that could provide additional insights into MS pathophysiology; the direction of the association is being explored.
Presenter Of 1 Presentation
LB1250 - Neuronal integrity is associated to peripheral leukocyte dysfunction in asymptomatic Multiple Sclerosis (MS) first-degree relatives (ID 2136)
Abstract
Background
Genetic studies suggest that peripheral immune dysfunctions occurs first in the causal chain of events leading to MS. However, these changes are typically not appreciated when patients present with their first symptom or with an asymptomatic inflammatory lesion; we have only sparsely characterized individuals before they develop disease. Thus, the earliest molecular events leading to damage of the CNS parenchyma remain unknown.
Objectives
To investigate the correlation between a marker of neuronal integrity (Neurofilament light chain, NfL) and the transcriptional profile of peripheral immune cells during the pre-symptomatic phase in a cohort of first-degree family members of MS patients.
Methods
Blood samples were collected from 124 asymptomatic first-degree relatives of MS patients part of the Genes & Environment in MS (GEMS) study, 7 MS patients, and 11 patients with other neuroimmune diseases (ONID), age (20-54). Serum samples were used to assess NfL levels using the SIMOA platform. Cryopreserved peripheral blood mononuclear cells (PBMC) were used to generate RNA-sequencing data. ‘CIBERSORT’ was used to estimate the proportions of major PBMC cell types (CD4 and CD8 T, monocyte, B, and NK) from the gene expression data. ‘WGCNA’ was used to to identify PBMC co-expressed gene modules. Available genotyping data were used to calculate a polygenic MS risk score for each individual.
Results
Serum NfL levels from GEMS individuals increased with age (p=2E-14), and were significantly lower than MS and ONID patients, adjusting for the effects of age and sex. NfL was not elevated in high-risk (high polygenic score) vs. low-risk family member subgroups.
After batch variation and technical variable correction 114 GEMS subjects qualified for PBMC bulk RNA-sequencing analysis. ‘WGCNA’ identified 37 co-expressed gene modules, 4 of which were associated with NfL levels (FDR-adjusted p<0.05), adjusting for effects of age, sex and estimated cell type proportions. One particular module is enriched in myeloid cells, and myeloid-mediated immunity and myeloid activation pathways; an association that persisted after accounting for the proportion of different cell types. The other 3 modules were all enriched for genes upregulated in chronic myelogenous leukemia and downregulated gene targets of KLF1 transcription factor.
Conclusions
The transcriptional programs of some peripheral immune cells strongly correlate with NfL levels; ongoing data replication. These results implicate a cross-talk between the peripheral immune system and the CNS that could provide additional insights into MS pathophysiology; the direction of the association is being explored.