Article Figures & Data
Figures
- Figure 1 Fingolimod Therapy Elevates Subnormal CD8+CD28− Treg in Therapy-Naive MS to Above HC Levels
(A) CD8+CD28− Treg as percent of lymphocytes were lower in therapy-naive stable patients with MS (3.68 ± 0.87%: p = 0.04) than in HCs (8.30 ± 1.95%) and even lower in therapy-naive exacerbating MS (1.47 ± 0.35%: p = 0.001 vs HC). Fingolimod therapy increased Treg to supranormal levels in stable MS (17.69 ± 3.10%: p = 0.002 vs therapy-naive stable) and maintained Treg at normal levels during attacks (6.52 ± 1.30%: p = 0.00004). (B) CD8+CD28+ CTL as percent of lymphocytes was greater in therapy-naive stable MS (14.06 ± 1.76%) than in fingolimod-treated stable patients (5.57 ± 1.20%: p = 0.0003). Similarly, CTLs were 12.27 ± 1.00% in therapy-naive exacerbating patients and only 5.34 ± 0.54% in fingolimod-treated exacerbating patients (p = 0.001). CTLs in HCs (11.33 ± 1.64%) were within the range of the therapy-naive stable and active MS groups. (C) Compared with HCs, the CD8+ Treg:CTL ratio was low in therapy-naive stable MS (ratio = 0.29; 0.35x of HC = 0.83) and in exacerbating patients (ratio = 0.12, 0.16x of HC). The subnormal CD8+ Treg:CTL ratio was dramatically reversed by fingolimod in stable MS (ratio = 6.52, 7.86x of HC) and in exacerbating MS (ratio = 1.31, 1.58x of HC). Flow cytometry of lymphocyte subsets. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Abbreviations: CTL = Cytolytic T lymphocyte; HC = healthy control; Treg = regulatory T cell.
- Figure 2 Fingolimod Therapy Decreases the Absolute Number of CD8+CD28+ CTL but Increases CD8+CD28− Treg In Vivo
(A) Absolute Treg numbers were 60 cells per microliter (µL) of blood in therapy-naive stable patients with MS (MS-s) and 83 cells/uL in stable fingolimod-treated patients (NS), but fell to 25 cells/µL with exacerbations (MS-a). Absolute CD8+ CTL counts decreased from 303 cells/µL blood in therapy-naive stable MS to 28 cells/µL in fingolimod-treated stable patients (p = 0.00005) and from 323 cells/uL in therapy-naive exacerbating MS to 18 cells/uL in fingolimod-treated exacerbating patients (p = 0.01). Absolute numbers were obtained by measuring the Treg:CTL as a % of the lymphocytes from flow cytometry and multiplying this percent by the absolute lymphocyte counts obtained from clinic visits. (B) The ratio of CD8+ Treg to CD8+ CTL is low in therapy-naive MS (therapy-naive stable MS = 0.4 and therapy-naive active MS = 0.3) but is increased 16.8-fold in fingolimod-treated stable patients and 16.7-fold in fingolimod-treated active patients. ∼ = 0.06, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Abbreviations: CTL = Cytolytic T lymphocyte; Treg = regulatory T cell.
- Figure 3 ConA Activation Increases CD69 Expression on CD8+CD28+ CTL More Than on CD8+CD28− Treg in HCs and Therapy-Naive RRMS
(A) After ConA activation of HC PBMCs, the median fluorescence intensity of CD69 expression on CD8+CD28+ CTL was 4–5-fold more than on CD8+CD28− Treg, across all conditions: media control, fingolimod, the FTY720 agonists, AAL-R, and FTY-P. (B) After ConA activation of therapy-naive, exacerbating RRMS (MS-a) PBMCs, CD69 expression on CTL and Treg in across all conditions was similar to activated HC cells. However, ConA activation decreased the number of CD8+CD28− Treg in MS compared with HCs. In unstimulated lymphocytes, CD69 expression on CD8+CD28 ± cells was minimal across all conditions and groups (0.2–4%; data not shown). Representative figure of 17 experiments with stable and active MS, see Supplemental eFigure 2 (links.lww.com/NXI/A787) for gating procedure. All CD28 ± cells are CD8+ and CD69+. Abbreviations: ConA = concanavalin A mitogen; CTL = Cytolytic T lymphocyte; HC = healthy control; PBMC = peripheral blood mononuclear cell; FTY-p = FTY-phosphate; Treg = regulatory T cell.
- Figure 4 In Vitro Immune Activation Depletes CD8+CD28− Treg
(A) The percentage of CD8+CD28− Treg in resting PBMCs was lower in therapy-naive patients with MS than in HCs (HC vs stable MS [MS-s], p = 0.04; HC vs exacerbating MS [MS-a], p = 0.003, unpaired t tests). Fingolimod-treated patients had higher Treg levels than therapy-naive MS (MS-s, p = 0.01; MS-a, p = 0.001), equivalent to levels in HCs. ConA activation decreased the percent of Treg in HCs (p = 0.05 vs resting/media controls; paired t tests). Activation tended to reduce the already low Treg population in therapy-naive stable (p < 0.11) and significantly reduced the percentage of Treg in exacerbating MS (p = 0.05). After activation, fingolimod-treated MS Treg levels drop (p = 0.04) to levels of HCs. (B) The percentage of CD8+CD28+ CTL in resting PBMCs tended to be lower in fingolimod-treated patients than in HCs (p = 0.06). ConA activation in fingolimod-treated patients further decreased CTL levels (p = 0.00005 vs activated HCs cells). ConA-activated PBMCs from therapy-naive stable MS and active MS had greater CTL levels than fingolimod-treated patients (p = 0.001 stable and p = 0.00003 active patients). The trend for a fall in CTLs after ConA activation in fingolimod-treated MS was nonsignificant. ∼ = 0.06, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Abbreviations: ConA = concanavalin A mitogen; CTL = Cytolytic T lymphocyte; HC = healthy control; PBMC = peripheral blood mononuclear cell; Treg = regulatory T cell.
- Figure 5 Age and MS Affect the Increase of CD8+CD28− Treg by Fingolimod Therapy
(A) The percentage of CD8+CD28− Treg in the resting lymphocyte population trended 2-fold higher in healthy older than in younger subjects (p = 0.09). The percentage of Treg in unstimulated older therapy-naive MS was lower than in unstimulated older HCs (p = 0.001) and did not increase with aging. The percentage of Treg in unstimulated fingolimod-treated younger patients was 5.8-fold higher than in unstimulated younger therapy-naive MS (p = 0.005) and 5.6-fold higher in unstimulated fingolimod-treated older patients than in older unstimulated therapy-naive MS (p = 0.001). On activation with ConA, Treg percentages did not significantly change in young and old HCs or in therapy-naive patients and tended to lose some of the Treg increase because of fingolimod therapy, although levels were still greater than in therapy-naive MS (p = 0.0002 for young and p = 0.03 for old). Stable and active patients with MS had similar profiles and were combined for analysis. (B) The percentage of CD8+CD28+ CTL within total lymphocytes in older HCs and in older therapy-naive MS was lower than that of younger subjects and did not change with ConA activation. However, fingolimod therapy decreased the percentage of CTL in unstimulated young (p = 0.00004) and old MS (p = 0.01) and in ConA-activated young (p = 0.02) and old MS (p = 0.01). Hashing indicates ConA activation. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Abbreviations: ConA = concanavalin A mitogen; CTL = Cytolytic T lymphocyte; HC = healthy control; Treg = regulatory T cell.
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