Basophils As a Potential Marker of Lupus Nephritis By Flow Cytometry

Figures & data

Figure 1. Flow cytometry panel from human peripheral blood.

After obtaining the sample, the red cells underwent lysis. The 14-color panel was performed on whole blood cells. Subpopulations of peripheral immune mononuclear cells (T cells, CD4⁺ T cells, CD8⁺ T cells, CD4⁻CD8⁻ T cells, CD4⁺CD8⁺ T cells, Tregs, B cells, NK cells, ILCs, basophils, MyDCs, pDCs, monocytes, classical [CD14⁺CD16⁻], intermediate [CD14⁺CD16^low] and nonclassical [CD14⁺CD16^hi] monocytes) were identified. The cellular gating depicted is representative of one healthy individual sample.

DC: Dendritic cell; FSC-A: Forward scatter area; FSC-H: Forward scatter height; HLA-DR: Human leukocyte antigen DR; ILC: Innate lymphoid cell; MyDC: Myeloid dendritic cell; NK: Natural killer; pDC: Plasmacytoid dendritic cell; SSC: Side scatter; Treg: Regulatory T cell; WBC: White blood cell.

Table 1. Optimized combination of 14 commercially available fluorochrome-conjugated antibodies to reliably quantify 18 subpopulations of immune cells.

Conjugation	Antibody	Clone (company)	Titer
FITC	Anti-human CD45 antibody	HI30 (BioLegend)	1:50
Percp-cy5.5	Anti-human CD14 antibody	M5E2 (BD)	1:50
BV510	Anti-human CD3 antibody	UCHT1 (BD)	1:50
Qdot655	Anti-human CD4 antibody	S3.5 (Invitrogen)	1:200
PE-CY7	Anti-human CD8 antibody	RPA-T8 (BD)	1:100
APC	Anti-human CD25 antibody	BC96 (BioLegend)	1:20
BUV395	Anti-human CD16 antibody	3G8 (BD)	1:100
BV605	Anti-human CD56 antibody	NCAM16.2	1:100
APC-CY7	Anti-human HLA-DR antibody	L243 (BioLegend)	1:100
BV421	Anti-human CD19 antibody	HIB19 (BD)	1:100
PE	Anti-human CD127 antibody	A019D5 (BioLegend)	1:25
BV710	Anti-human CD123 antibody	9F5 (BD)	1:50
Alexa Fluor^® 700	Anti-human CD11c antibody	Bu15 (BioLegend)	1:50
DAPI	Live/dead		1:50

Table 2. Demographic, clinical and serological features of patients with systemic lupus erythematosus.

Characteristic	SLE (n = 60)	HC (n = 20)	p-value
Age (years)	35.27 ± 12.21	30.90 ± 8.58	0.09
Sex, n (% female)	49 (81.67)	15 (75)	0.53
Disease duration (years)	5.09 ± 0.92
Current concomitant medications	58 (96.67)
Hydroxychloroquine, n (%)	36 (60)
Prednisone, n (%)	58 (96.67)
Current dose of prednisone (mg/day)	33.67 ± 25.53
Cyclophosphamide, n (%)	20 (33.33)
Cumulate dose of cyclophosphamide (g)	2.25 ± 2.16
Mycophenolate mofetil	3 (5)
Leflunomide	1 (1.67)
SLEDAI-2000	6.2 ± 4.46
Active SLE patients (SLEDAI-2000 ≥5)	32 (53.33)
Mean SLEDAI-2000 scores	9.53 ± 3.25
Inactive SLE patients (SLEDAI-2000 <5)	28 (46.67)
Mean SLEDAI-2000 scores	2.39 ± 1.77
System involved, n (% positive)
Mucocutaneous involvement	26 (43.33)
Arthritis	21 (35)
Nephritis	38 (63.33)
Hematological involvement	25 (41.67)
Laboratory features, n (% positive)
ANA	60 (100)
ANA titer <1:1000	27 (45)
ANA titer ≥1:1000	33 (55)
Anti-dsDNA antibody	34 (56.67)
Anti-dsDNA titer <1:100	21 (35)
Anti-dsDNA titer ≥1:100	13 (21.67)
Anti-SSA antibody	35 (58.33)
Anti-SSB antibody	21 (35)
Anti-Sm antibody	29 (48.33)
Anti-RNP antibody	35 (58.33)
C3 (g/l)	0.56 ± 0.04
C4 (g/l)	0.12 ± 0.01
IgG (g/l)	15.17 ± 9.41
IgA (mg/l)	2597.08 ± 1438.96
IgM (mg/l)	1134.98 ± 939.55
IgE (IU/ml)	47.06 ± 64.7
ESR (mm/h)	33.78 ± 20.8
CRP (mg/dl)	8.67 ± 15.45
Leukocyte count (10^9/l)	7.33 ± 0.61
Lymphocyte count (10^9/l)	1.29 ± 0.26
Granulocyte count (10^9/l)	5.50 ± 0.54

Data are expressed as absolute numbers (%) or the mean ± standard deviation.

ANA: Anti-nuclear antibody; C3/4: Complement component 3/4; ESR: Erythrocyte sedimentation rate; HC: Healthy control; SLE: Systemic lupus erythematosus; SLEDAI-2000: SLE Disease Activity Index 2000; Sm: Smith; SSA: Sjögren syndrome-related antigen A; SSB: Sjögren syndrome-related antigen B.

Table 3. Differences in immune cellular compositions between patients with systemic lupus erythematosus and healthy controls.

Variables	SLE (n = 60)	HC (n = 20)	p-value
			Univariate	Multivariate
B cells	17.41 ± 11.1	12.56 ± 4.02	NS
T cells	76.17 ± 11.87	73.96 ± 8.32	NS
CD4^+ T cells	43.59 ± 13.93	54.00 ± 8.18	<0.001	0.22
CD8^+ T cells	49.81 ± 14.53	36.61 ± 8.12	<0.001	0.22
CD4^−CD8^− T cells	5.12 ± 3.67	8.61 ± 4.94	<0.001	0.027
CD4^+CD8^+ T cells	1.47 ± 1.54	0.78 ± 0.34	0.028	0.379
Tregs	4.31 ± 2.00	6.18 ± 1.31	<0.001	0.095
Monocytes	1.48 ± 1.56	1.58 ± 1.14	NS
Classical monocytes (CD14^+CD16^−)	76.24 ± 14.84	80.33 ± 14.62	NS
Intermediate monocytes (CD14^+CD16^low)	11.41 ± 7.10	12.65 ± 9.25	NS
Nonclassical monocytes (CD14^+CD16^hi)	11.54 ± 12.16	6.64 ± 5.80	NS
NK cells	4.70 ± 3.97	11.85 ± 7.46	<0.001	<0.001
CD56^dim NK cells	85.18 ± 12.14	91.23 ± 6.46	NS
CD56^bright NK cells	14.82 ± 12.14	8.77 ± 6.46	NS
MyDCs	0.05 ± 0.05	0.10 ± 0.05	<0.001	0.571
pDCs	0.01 ± 0.01	0.05 ± 0.03	<0.001	0.092
Basophils	0.03 ± 0.09	0.12 ± 0.12	<0.001	0.649
ILCs	0.47 ± 0.38	0.94 ± 0.60	<0.001	0.014

Data of monocytes, MyDCs, pDCs and basophils are expressed as a percentage of live CD45⁺ white blood cells. Data for B cells, T cells, NK cells and ILCs are expressed as a percentage of live lymphocytes. Data are shown as the mean ± standard deviation. p < 0.05 indicates statistical significance.

HC: Healthy control; ILC: Innate lymphoid cell; MyDC: Myeloid dendritic cell; NK: Natural killer; NS: Not significant; pDC: Plasmacytoid dendritic cell; SLE: Systemic lupus erythematosus.

Table 4. Differences in immune cellular compositions among patients with active systemic lupus erythematosus, patients with inactive systemic lupus erythematosus and healthy controls.

Variables	Active SLE (n = 32)	Inactive SLE (n = 28)	HC (n = 20)	p-value ^†		p-value ^‡		p-value ^§
				Univariate	Multivariate	Univariate	Multivariate	Univariate	Multivariate
B cells	17.37 ± 12.13	17.45 ± 10.01	12.56 ± 4.02	NS		NS		NS
T cells	76.37 ± 12.97	75.95 ± 10.7	73.96 ± 8.32	NS		NS		NS
CD4^+ T cells	38.36 ± 9.67	49.57 ± 15.72	54.00 ± 8.18	<0.01	0.65	<0.001	0.64	NS
CD8^+ T cells	55.69 ± 10.88	43.09 ± 15.42	36.61 ± 8.12	<0.01	0.19	<0.001	0.64	NS
CD4^−CD8^− T cells	4.2 ± 2.34	6.18 ± 4.58	8.61 ± 4.94	NS		<0.001	0.64	NS
CD4^+CD8^+ T cells	1.75 ± 1.9	1.16 ± 0.9	0.78 ± 0.34	NS		<0.05	0.66	NS
Tregs	4.62 ± 2.1	3.95 ± 1.86	6.18 ± 1.31	NS		<0.001	0.85	<0.001	0.15
Monocytes	1.85 ± 1.76	1.06 ± 1.19	1.58 ± 1.14	NS		NS		NS
Classical monocytes (CD14^+CD16^−)	76.28 ± 16.2	76.19 ± 13.42	80.33 ± 14.62	NS		NS		NS
Intermediate monocytes (CD14^+CD16^low)	10.38 ± 6.69	12.6 ± 7.48	12.65 ± 9.25	NS		NS		NS
Nonclassical monocytes (CD14^+CD16^hi)	12.69 ± 13.72	10.22 ± 10.17	6.64 ± 5.80	NS		NS		NS
NK	4.79 ± 4.38	4.6 ± 3.51	11.85 ± 7.46	NS		<0.001	0.15	<0.001	0.07
CD56^dim NK cells	86.1 ± 13.22	84.13 ± 10.91	91.23 ± 6.46	NS		NS		NS
CD56^bright NK cells	13.9 ± 13.22	15.87 ± 10.91	8.77 ± 6.46	NS		NS		NS
MyDCs	0.05 ± 0.06	0.04 ± 0.04	0.10 ± 0.05	NS		<0.001	0.51	<0.001	0.16
pDCs	0.01 ± 0.01	0.02 ± 0.02	0.05 ± 0.03	NS		<0.001	0.53	<0.001	0.83
Basophils	0.03 ± 0.06	0.04 ± 0.12	0.12 ± 0.12	NS		<0.001	0.81	<0.01	0.54
CD127^+ ILCs	0.43 ± 0.33	0.51 ± 0.44	0.94 ± 0.60	NS		<0.001	0.79	<0.01	0.23

Data for monocytes, MyDCs, pDCs and basophils are expressed as a percentage of live CD45⁺ white blood cells. Data for B cells, T cells, NK cells and ILCs are expressed as a percentage of live lymphocytes. Data for CD4⁺ T cells, CD8⁺ T cells, CD4⁺CD8⁺ T cells, CD4⁻CD8⁻ T cells, Tregs, classical/intermediate/nonclassical monocytes, CD56^dim NK cells and CD56^bright NK cells are expressed as a percentage of the parent cells. Data are shown as the mean ± standard deviation. p < 0.05 indicates statistical significance.

† active SLE versus inactive SLE.

‡ active SLE versus HC.

§ inactive SLE versus HC.

HC: Healthy control; ILC: Innate lymphoid cell; MyDC: Myeloid dendritic cell; NK: Natural killer; NS: Not significant; pDC: Plasmacytoid dendritic cell; SLE: Systemic lupus erythematosus.

Figure 2. Differences in basophils between patients with and without lupus nephritis.

The proportion of basophils was decreased in patients with nephritis (n = 38) compared with patients without nephritis (n = 22). Data are shown as the mean ± standard deviation. p < 0.05 indicates statistical significance.