Evaluation of Age-Dependent Immune Signatures in Patients With Multiple Sclerosis

Abstract

Background and objectives: In MS, an age-related decline in disease activity and a decreased efficacy of disease-modifying treatment have been linked to immunosenescence, a state of cellular dysfunction associated with chronic inflammation.

Methods: To evaluate age-related immunologic alterations in MS, we compared immune signatures in peripheral blood (PB) and CSF by flow cytometry in patients with relapsing-remitting (RR) (PB n = 38; CSF n = 51) and primary progressive (PP) MS (PB n = 40; CSF n = 36) and respective controls (PB n = 40; CSF n = 85).

Results: Analysis revealed significant age-related changes in blood immune cell composition, especially in the CD8 T-cell compartment of healthy donors (HDs) and patients with MS. However, HDs displayed a strong age-dependent decline in the expression of the immunoregulatory molecules KLRG1, LAG3, and CTLA-4 on memory CD8 T cells, whereas this age-dependent reduction was completely abrogated in patients with MS. An age-dependent increase in the expression of the costimulatory molecule CD226 on memory CD8 T cells was absent in patients with MS. CD226 expression correlated with disability in younger (≤50 years) patients with MS. CSF analysis revealed a significant age-dependent decline in various immune cell populations in PPMS but not RRMS, suggesting a differential effect of aging on the intrathecal compartment in PPMS.

Discussion: Our data illustrate that aging in MS is associated with a dysbalance between costimulatory and immunoregulatory signals provided by CD8 T cells favoring a proinflammatory phenotype and, more importantly, a pattern of premature immune aging in the CD8 T-cell compartment of young patients with MS with potential implications for disease severity.

Figure 1. Basic Immune Cell Subset Composition in Young and Old Patients With MS and HDs

Immune cell subset composition in the peripheral blood of young (≤50 years) and old (>50 years) patients with multiple sclerosis (MS) (young: n = 40, old: n = 38) and HDs (young: n = 20, old: n = 20). Demographic data of study subjects are depicted in eTable 1 (A–D, links.lww.com/NXI/A620). Frequencies of lymphocytes (A), B cells (B), CD4 (C), and CD8 (D) T cells. Data are displayed as boxplots of the median and the 25th and 75th percentile ± interquartile range. Statistical analysis was conducted by the 2-tailed Mann-Whitney test. Differences were considered statistically significant with the following p values: *p < 0.05, **p < 0.01, and ***p < 0.001. HD = healthy donor.

Figure 2. Age-Related Changes in the CD8 T-Cell Compartment in Patients With MS and HDs

Composition of the CD8 T-cell compartment in the peripheral blood of young (≤50 years) and old (>50 years) patients with MS (young: n = 40, old: n = 38) and HDs (young: n = 20, old: n = 20). Demographic data of study subjects are depicted in eTable 1 (A–D, links.lww.com/NXI/A620). Proportions of naive (A), memory (B), effector memory (C), and central memory (D) CD8 T cells. Data are displayed as boxplots of the median and the 25th and 75th percentile ± interquartile range. Statistical analysis was conducted by the 2-tailed Mann-Whitney test. Differences were considered statistically significant with the following P values: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. CM = central memory; EM = effector memory; HD = healthy donor.

Figure 3. Immunoregulatory Markers on T Cells in Young and Old Patients With MS and HDs

Flow cytometric analysis of frozen peripheral blood mononuclear cells from young (≤50 years) and old (>50 years) patients with MS (MS: young: n = 40, old: n = 38; RRMS: young: n = 20, old: n = 18; PPMS: young: n = 20, old: n = 20) and HDs (young: n = 20, old: n = 20). Demographic data of study subjects are depicted in eTable 1, links.lww.com/NXI/A620. (A) MFI of KLRG1 on memory CD8 T cells of patients with MS and HDs. (B) Correlation analysis of MFI of KLRG1 on memory CD8 T cells with age of HDs (n = 40), patients with RRMS (n = 38), and patients with PPMS (n = 40). (C) MFI of LAG3 on memory CD8 T cells of patients with MS and HDs. (D) Correlation analysis of MFI of LAG3 on memory CD8 T cells with age of HDs (n = 40), patients with RRMS (n = 38), and patients with PPMS (n = 40). Data are displayed as boxplots of the median and the 25th and 75th percentile ± interquartile range. Statistical analysis was conducted by the 2-tailed Mann-Whitney test. For correlation analysis, the Pearson product-moment correlation coefficients (Pearson R) were computed. Differences were considered statistically significant with the following p values: **p < 0.01 and ***p < 0.001. HD = healthy donor; KLRG1 = killer cell lectin-like receptor subfamily G member 1; LAG3 = lymphocyte-activation gene 3; MFI = mean fluorescence intensity; MS = multiple sclerosis; PPMS = primary progressive MS; RRMS = relapsing-remitting MS.

Figure 4. Age-Related Expression of the Costimulatory Molecule CD226 on T Cells in Patients With MS and HDs

Flow cytometric analysis of frozen peripheral blood mononuclear cells from young (≤50 years) and old (>50 years) patients with MS (MS: young: n = 40, old: n = 38; RRMS: young: n = 20, old: n = 18; PPMS: young: n = 20, old: n = 20) and HDs (young: n = 20, old: n = 20). Demographic data of study subjects are depicted in eTable 1, links.lww.com/NXI/A620. (A) MFI of CD226 (DNAM-1) on memory CD8 T cells. (B) Correlation analysis of MFI of CD226 on memory CD8 T cells with age of HDs (n = 40), patients with RRMS (n = 38), and patients with PPMS (n = 40). (C, D) Correlation analysis of CD226 expression on memory CD8 T cells with the EDSS score of young (C) and old (D) patients with MS. Data are displayed as boxplots of the median and the 25th and 75th percentile ± interquartile range. Statistical analysis was conducted by the 2-tailed Mann-Whitney test. For correlation analysis, the Pearson product-moment correlation coefficients (Pearson R) were computed. Differences were considered statistically significant with the following p values: **p < 0.01 and ***p < 0.001. EDSS = Expanded Disability Status Scale; HD = healthy donor; MS = multiple sclerosis; MFI = mean fluorescence intensity; PPMS = primary progressive MS; RRMS = relapsing-remitting MS.

Figure 5. Age-Dependent Immune Cell Subset Composition in the CSF of Patients With MS and Noninflammatory Controls

Immune cell subset composition in the in the CSF of young (≤50 years) and old (>50 years) NICs (young n = 60; old: n = 25) and patients with RRMS (young: n = 41; old: n = 10) and PPMS (young: n = 15; old: n = 21). Demographic data of study subjects are depicted in eTable 2, links.lww.com/NXI/A621. (A–D) Absolute numbers of lymphocytes (A), B cells (B), T cells (C), and plasma cells (D). Data are displayed as boxplots of the median and the 25th and 75th percentile ± interquartile range. Statistical analysis was conducted by the 2-tailed Mann-Whitney test. Differences were considered statistically significant with the following p-values: **p < 0.01 and ***p < 0.001. NIC = noninflammatory control; PPMS = primary progressive MS; RRMS = relapsing-remitting MS.