The association between neovascular age-related macular degeneration and regulatory T cells in peripheral blood

Figures & data

Figure 1 Flow cytometric identification of regulatory T cells (Tregs), nTregs (naïve Tregs) and RTE-Tregs (recent thymic emigrant Tregs) in the peripheral blood of a patient with AMD. Lymphocytes were identified and gated on a forward/side-scatter plot (A). CD4⁺ lymphocytes were then identified and gated on a CD4/F8 plot (B), and the percentage of CD4⁺ cells within the lymphocyte population was measured. The CD25^highCD127^low Treg population was identified and gated on a CD25/CD127 plot (C), and the percentage of Tregs within the CD4⁺ population was measured. Fluorochrome-matched negative isotype controls (IgG2b and IgG1) were used to set a gate containing 1% of the events, and the gates were transferred to a CD45RA histogram (D) and a CD31 histogram (E). The percentages of CD45RA⁺-naïve Tregs and CD31⁺ RTE-Tregs within the Treg population were measured. The letters A–D in figures 1D and 1E mark the gates containing 1% of the events.

Abbreviations: IgG, immunoglobulin G; FS, forward-scatter; SS, side-scatter; AMD, age-related macular degeneration.

Table 1 Demographics and clinical characteristics of study participants with neovascular age-related macular degeneration and healthy controls

Demographics and clinical characteristics	Neovascular age-related macular degeneration	Controls	P-value
	(n=21)	(n=12)
Median age (interquartile range), yrs	82 (78–86)	75 (72–79)	0.012 (Mann–Whitney U-test)
Sex, n (%)			0.895 (chi-square test)
Male	10 (47.6)	6 (50.0)
Female	11 (52.4)	6 (50.0)
Smoking history, n (%)			0.947 (chi-square test)
Never smoker	9 (42.9)	5 (41.7)
Ever smoker	12 (57.1)	7 (58.3)
Risk factors
Alcohol consumption, units/week, median (interquartile range)	4 (0–7)	5 (1–10)	0.840 (Mann–Whitney U-test)
BMI, kg/m^2, median (interquartile range)	26 (24–27)	25 (22–28)	0.895 (Mann–Whitney U-test)
Physically active, n (%)	10 (47.6)	8 (66.7)	0.290 (chi-square test)
Cardiovascular disease, n (%)	5 (23.8)	4 (33.3)	0.555 (chi-square test)
Hypertension, n (%)	11 (52.4)	8 (66.7)	0.424 (chi-square test)
Dyslipidemia, n (%)	7 (33.3)	1 (8.3)	0.107 (chi-square test)
Markers of inflammation
Serum C-reactive protein, mg/L, median (interquartile range)	2.9 (2.9–2.9)	2.9 (2.9–3.0)	0.687 (Mann–Whitney U-test)
White blood cell count, 10^9/L, mean (SD)	6.0 (1.3)	5.6 (1.3)	0.456 (independent samples t-test)

Abbreviations: yrs, years; SD, standard deviation; BMI, body mass index.

Figure 2 Percentages of CD4⁺ lymphocytes, CD25^highCD127^low Tregs, CD45RA⁺-naïve Tregs, and CD31⁺ RTE-Tregs in the peripheral blood of patients with neovascular age-related macular degeneration and healthy controls.

Notes: A and B; Bars – means, whiskers – SD; C, D, E and F: Dots = medians, whiskers = interquartile ranges.

Abbreviations: AMD, age-related macular degeneration; SD, standard deviation; Tregs, regulatory T cells; RTE-Tregs, recent thymic emigrant Tregs.

Table 2 Percentages of lymphocytes in the peripheral blood of patients with neovascular age-related macular degeneration and healthy controls

Cell population	Neovascular age-related macular degeneration	Controls	P-value
CD4, mean (SD)a	43.53 (13.37)	39.7 (8.54)	0.379 (independent samples t-test)
CD25^highCD127^low Tregs, mean (SD)b	5.71 (1.80)	6.09 (1.71)	0.554 (independent samples t-test)
CD45RA^+ naïve Tregs, median (interquartile range)c	17.28 (14.04–23.20)	14.38 (11.37–15.93)	0.188 (Mann–Whitney U-test)
CD31^+ RTE-Tregs, median (interquartile range)d	2.82 (2.28–4.07)	2.63 (1.18–4.05)	0.895 (Mann–Whitney U-test)
CD45RA^+ naïve Tregs, median (interquartile range)e	1.09 (0.62–1.41)	0.87 (0.58–1.21)	0.349 (Mann–Whitney U-test)
CD31^+ RTE-Tregs, median (interquartile range)f	0.16 (0.13–0.27)	0.14 (0.09–0.23)	0.454 (Mann–Whitney U-test)

Notes:

a Percentage of CD4⁺ cells in the lymphocyte population.

b Percentage of CD25^highCD127^low Tregs in the CD4⁺ population.

c Percentage of CD45RA⁺ naïve Tregs in the Treg population.

d Percentage of CD31⁺ RTE-Tregs in the Treg population.

e Percentage of CD45RA⁺ naïve Tregs in the CD4⁺ population.

f Percentage of CD31⁺ RTE-Tregs in the CD4⁺ population.

Abbreviations: SD, standard deviation; Tregs, regulatory T cells; RTE-Tregs, recent thymic emigrant Tregs.