A new approach for evaluating antigen-specific T cell responses to myelin antigens during the course of multiple sclerosis

Abstract

We used a flow cytometry assay to measure proliferation and cytokine production of self-antigen-specific T cells in individual patients during the clinical course of multiple sclerosis (MS). Myelin-associated oligodendrocytic basic protein (MOBP) was selected for proof of principles in the assay, along with myelin basic protein (MBP) to assess specific activated T cells in 10 MS patients over an 18-month period, in parallel with brain magnetic resonance imaging (MRI) scans and clinical rating scale. A positive correlation occurred between antigen-specific T cell proliferation and interferon-gamma production with clinical relapses and MRI lesion activity that was absent when the same patients were in remission.

Fig. 1

Proliferation and cytokine production by CD4⁺ T cells from patient 3 after myelin antigen stimulation. CFSE-labeled human PBMC from patient 3 were incubated either with or without different myelin antigens for 10 days and then stimulated for 5 h in the presence of PMA, ionomycin and brefeldin A before labeling for surface CD4⁺ T cells and intracellular IFN-γ. Dot plots from the flow cytometry assays represent gated cells for CD4⁺ lymphocytes. The antigen added to the culture is written underneath each dot plot: Nil: no antigen, M1: MOBP aa 1–19, M11: MOBP aa 11–29, M20: MOBP aa 20–38, M31: MOBP aa 31–49, M41: MOBP aa 41–59, MBP: myelin basic protein. Percentage of cells that proliferated is indicated in appropriate quadrant either IFN-γ+ (upper left quadrant) or IFN-γ − cells (lower left quadrant). (A) Cells taken at the 7th month of the follow-up; (B) cells taken at the 12th month of the follow-up.

Fig. 2

Proliferation and cytokine production by CD4⁺ T cells from patient 5 after myelin antigen stimulation. (A) Cells taken at the 10th month of the follow up; (B) cells taken at the 18th month of the follow-up.

Fig. 3

Clinical data and myelin reactivity of T cells from patient 1. At the time points indicated, patient 1 was examined for EDSS and for gadolinium-enhanced brain MRI scans. Simultaneously, this patient gave blood from which PBMC were isolated and frozen for later analysis. Arrows labeled 1, 2, 3, 4 and 5 indicate times when CFSE-labeled PBMC were cultured for 10 days with and without different myelin antigens specified. Supernatants were harvested for ELISA, then cells were stimulated for 5 h with the combined PMA, ionomycin and brefeldin A before labeling for surface CD4 and intracellular IFN-γ. (A) Clinical data from donor: EDSS (■, solid line) and gadolinium-enhanced brain MRI lesion (○ dashed line). (B) Amount of IFN-γ over the background measured by ELISA in the supernatant after 10-day culture with different myelin antigens M1: MOBP aa 1–19, M11: MOBP aa 11–29, M20: MOBP aa 20–38, M31: MOBP aa 31–49, M41: MOBP aa 41–59. (C) Percentage of CD4⁺ IFN-γ-producing T cells that proliferated over the background after culture with different myelin antigens and PMA, ionomycin and brefeldin A stimulation (time points are indicated by the arrows on panel A). (D) Percentage of CD4⁺ T cells over the background that proliferated after culture with different myelin antigens (time points are indicated by the arrows on panel A).

Fig. 4

Clinical data and myelin-reactivity of T cells from patients 2, 3 and 4. Patient 2: (A) clinical data EDSS (■, solid line) and gadolinium-enhanced brain MRI lesion (○ dashed line). (B) Percentage of CD4⁺ T cells over the background that proliferated and produced IFN-γ after the culture with different myelin antigens for the time points indicated by the arrows on panel A. Patient 3: (C) clinical data EDSS and GD+ brain lesion. (D) Percentage of CD4⁺ T cells over the background that proliferated after culture with different myelin antigens (time points are indicated by the arrows on panel C). Patient 4: (E) clinical data EDSS and GD+ brain lesion. (F) Percentage of CD4⁺ T cells over the background that proliferated after culture with different myelin antigens (time points are indicated by the arrows on panel E).

Fig. 5

Clinical data and myelin reactivity of T cells from patients 5, 6 and 7. Patient 5: (A) clinical data EDSS (■, solid line) and gadolinium-enhanced brain MRI lesion (○ dashed line). (B) Percentage of CD4⁺ T cells over the background that proliferated and produced IFN-γ after the culture with different myelin antigens for the time points indicated by the arrows on panel A. Patient 6: (C) clinical data EDSS and GD+ brain lesion. (D) Percentage of CD4⁺ T cells over the background that proliferated after culture with different myelin antigens (time points are indicated by the arrows on panel C). Patient 7: (E) clinical data EDSS and GD+ brain lesion. (F) Percentage of CD4⁺ T cells over the background that proliferated after culture with different myelin antigens (time points are indicated by the arrows on panel E).

Fig. 6

Clinical data and myelin reactivity of T cells from patients 8, 9 and 10. Patient 8: (A) clinical data EDSS (■, solid line) and gadolinium-enhanced brain MRI lesion (○ dashed line). (B) Percentage of CD4⁺ T cells over the background that proliferated and produced IFN-γ after the culture with different myelin antigens for the time points indicated by the arrows on panel A. Patient 9: (C) clinical data EDSS and GD+ brain lesion. (D) Percentage of CD4⁺ T cells over the background that proliferated after culture with different myelin antigens (time points are indicated by the arrows on panel C). Patient 10: (E) clinical data EDSS and GD+ brain lesion. (F) Percentage of CD4⁺ T cells over the background that proliferated after culture with different myelin antigens (time points are indicated by the arrows on panel E).