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i e
Table 1.
1 Clinical characteristics of 25 patients with autoimmune neutropenia
Human neutrophil
Pat
Patient
Age at onset
Initial manifestation for the diagnosis of
antigens against
(months)
neutropenia
antineutrophil
Sex*
num
number
antibodies
Pyoderma
1a
10
Acute upper respiratory infection
1a
12
Pyrexia
1a
Lymphadenitis
1a/1b
14
Pyrexia
1a
22
Pyoderma
1a
17
Adeno Virus infection
1a
Pyrexia
1a/1b
22
Acute upper respiratory infection
1a
10
14
Lymphadenitis
1a
11
38
Pyrexia
1a
12
12
Pyrexia
1a
13
Pyrexia
1a/1b
14
Pyoderma
1a/1b
15
Pyrexia
1a/1b
16
Not doing well
1a
17
21
Acute bronchitis
1a/1b
18
10
Pyrexia
1a/1b
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Acute upper respiratory infection
1a
20
11
Pyrexia
1a
21
Acute bronchitis
1a
22
Respiratory syncytial virus infection
1a
23
10
Lymphadenitis
1a
24
31
Acute bronchitis
1a
25
20
Pyrexia
1a
Accepted Article
rticle
19
*M
M, male;
ma F, female
Patient examined for both the frequency of total/activated regulatory T cells and T cell receptor (TCR)-Vβ
Patien
repertoi
rep
repertoire.
Patients 18–25: analysis of frequency of total and activated regulatory T cells.
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Accepted Article
Table 2.
2 Standard value of TCR-Vβ family usage in CD4⁺⁺ T cells
β fam
Vβ
family
%Mean
95%CI
SD
%Median
IQR
β1
Vβ
3.08
2.93, 3.24
0.35
3.09
0.54
β2
Vβ
10.84
10.1, 11.6
1.66
11.10
2.46
β3
Vβ
6.75
5.78, 7.72
2.19
7.13
2.79
β4
Vβ
2.10
2.01, 2.19
0.20
2.08
0.47
Vβ 5.1
4.73
4.10, 5.36
1.43
5.00
1.22
Vβ
β 5.2
0.98
0.93, 1.03
0.12
0.97
0.20
Vβ
β 5.3
0.86
0.82, 0.90
0.08
0.88
0.12
Vβ
β 7.1
2.06
1.88, 2.24
0.40
2.05
0.57
Vβ
β 7.2
0.40
0.20, 0.59
0.44
0.07
0.75
Vβ
β8
3.44
3.16, 3.72
0.63
3.46
0.88
Vβ
β9
3.12
2.94, 3.30
0.41
3.17
0.60
Vβ
β 11
0.79
0.73, 0.84
0.13
0.79
0.20
Vβ
β 12
1.78
1.59, 1.96
0.43
1.72
0.67
Vβ
β 13.1
4.07
3.68, 4.45
0.88
4.13
1.32
Vβ
β 13.2
1.84
1.46, 2.21
0.84
1.60
1.33
Vβ
β 13.6
1.78
1.70, 1.86
0.18
1.76
0.25
Vβ
β 14
2.70
2.53, 2.86
0.37
2.69
0.46
Vβ
β 16
0.83
0.77, 0.89
0.13
0.84
0.17
Vβ
β 17
5.20
4.96, 5.45
0.55
5.27
0.86
Vβ
β 18
2.00
1.87, 2.13
0.29
2.06
0.39
Vβ
β 20
3.27
2.87, 3.66
0.89
3.34
1.48
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1.54
1.41, 1.66
0.28
1.55
0.33
Vβ
β 22
3.80
3.57, 4.03
0.52
3.78
0.67
Vβ
β 23
0.46
0.42, 0.49
0.08
0.43
0.12
Accepted Article
Vβ
β 21.3
TCR, T cell receptor; 95%CI, 95% confidential interval; SD, standard deviation; IQR, interquartile range (75th–
TC
25th
5t per
percentile).
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Accepted Article
ic
Table 33. Standard value of TCR-Vβ family usage in CD4⁺⁺CD25− T cells and CD4+CD25+CD127low T cells
Regulatory T cell
Conventional T cells
(CD4+CD25−T cells)
(CD4+CD25+CD127low T cells)
Vβ
%Mean
95%CI
SD
%Median IQR
%Mean
95%CI
SD
Vβ 1
3.06
2.89, 3.24
0.40
3.11
Vβ 2
10.80
10.0, 11.6
1.70
Vβ 3
6.85
5.86, 7.83
Vβ 4
2.10
Vβ 5.1
%Median IQR
0.62
2.92
2.77, 3.07
0.34
3.01
0.36
10.70
2.74
10.9
10.2, 11.7
1.65
11.10
2.44
2.20
7.28
2.77
5.74
4.87, 6.61
1.97
5.81
2.68
2.01, 2.19
0.04
2.09
0.32
2.00
1.85, 2.15
0.33
1.96
0.50
4.59
3.97, 5.21
1.40
4.92
1.23
6.03
5.21, 6.84
1.84
6.43
1.44
Vβ 5.2
0.97
0.92, 1.03
0.12
0.96
0.18
1.13
1.06, 1.20
0.16
1.12
0.22
Vβ 5.3
0.84
0.80, 0.88
0.08
0.84
0.11
1.03
0.95, 1.11
0.18
1.03
0.22
Vβ 7.1
2.04
1.85, 2.22
0.41
2.07
0.58
1.83
1.67, 1.99
0.35
1.88
0.60
Vβ
β 7.2
0.40
0.20, 0.59
0.44
0.07
0.76
0.40
0.23, 0.58
0.39
0.23
0.68
Vβ 8
3.47
3.18, 3.75
0.65
3.49
0.85
3.22
2.97, 3.46
0.55
3.25
0.75
Vβ 9
3.14
2.96, 3.32
0.41
3.19
0.64
2.83
2.65, 3.00
0.39
2.78
0.56
Vβ 11
0.78
0.72, 0.84
0.13
0.77
0.19
0.73
0.64, 0.81
0.19
0.76
0.29
Vβ 12
1.74
1.54, 1.94
0.44
1.66
0.63
2.26
2.05, 2.47
0.48
2.41
0.75
Vβ 13.1
4.12
3.73, 4.52
0.90
4.20
1.39
3.72
3.38, 4.06
0.76
3.94
1.05
Vβ
β 13.2
1.79
1.41, 2.16
0.85
1.51
1.33
1.98
1.59, 2.36
0.87
1.46
1.38
Vβ 13.6
1.75
1.67, 1.82
0.18
1.75
0.25
2.15
2.00, 2.30
0.34
2.16
0.46
Vβ 114
2.70
2.53, 2.87
0.38
2.65
0.52
2.74
2.55, 2.93
0.42
2.70
0.42
Vβ
β 16
0.83
0.77, 0.89
0.14
0.85
0.14
0.73
0.64, 0.81
0.19
0.73
0.31
family
fami
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5.21
4.96, 5.47
0.58
5.26
0.87
4.86
4.58, 5.12
0.61
4.87
1.21
Vβ 18
1.99
1.86, 2.13
0.30
2.02
0.41
1.99
1.85, 2.11
0.30
2.02
0.39
Vβ 20
3.37
2.95, 3.78
0.93
3.45
1.56
2.61
2.34, 2.89
0.63
2.65
0.80
Vβ
β 21.3
1.55
1.67, 1.42
0.28
1.57
0.33
1.49
1.38, 1.60
0.25
1.48
0.33
Vβ 22
3.83
3.59, 4.06
0.52
3.81
0.71
3.32
3.03, 3.61
0.65
3.28
1.16
Vβ
β 23
0.45
0.42, 0.48
0.07
0.43
0.12
0.41
0.35, 0.47
0.14
0.39
0.12
Accepted Article
Vβ 17
TCR,
CR, T cell receptor; 95%CI, 95% confidential interval; SD, standard deviation; IQR, interquartile range (75th–
25t
25th per
percentile).
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Accepted Article
Figure
1. Flow cytometric analysis of regulatory T cells (Tregs) and Treg subpopulations
CD4+CD
CD
CD25+FOXP3high T cells are counted as total Tregs and CD4+CD25+FOXP3highCD45RA− T cells in CD4+
T cells
((Fraction II in the Treg subpopulations) are counted as activated Tregs (a). Data are presented in
box-plo
box
box-plots which display the minimum value, 25th, 50th, 75th, maximum value, and describe means as X (b). For
of T cell receptor (TCR)-Vβ usage of each T cell subsets by flow cytometry, CD4+CD25− T cells
the evaluation
eval
in
n CD4
C + T cell are classified as conventional T cells (Tcons) and CD4+CD25+CD127low T cells are classified as
Tr
Tregs.
Each T cell subset IS sorted with fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated
anti-TC
ant
anti-TCR-Vβ family antibodies (c). The data for the percentage of each T cell subset (Tcons in CD4+ T cell,
Tre in CD4+ T cell) are presented as box-plots (d). *p = 0.0123, **p = 0.0123, ***p = 0.0113. n.s; not
Tregs
significa
significant. (Wilcoxon rank-sum test).
Figure 2. The usages of 24 TCR-Vβ families in CD4⁺⁺ T cells
Dat are represented as box-plots. Analysis was performed in the control group (Cnt, n=22) and autoimmune
Data
nneutrop
neutropenia (AIN) group (AIN, n = 17). *Vβ 9, p = 0.0252, **Vβ 17, p = 0.0361 (Wilcoxon rank-sum test).
Figure 3. The usages of 24 TCR- Vβ families in regulatory T cells (Tregs) (CD4+CD25+CD127low T cells)
Fig
an con
and
conventional T cells (Tcons) (CD4+CD25− T cells)
Data are represented as box-plots. Analysis was performed in the control group (Cnt, n = 22) and autoimmune
neutropenia (AIN) group, (AIN, n = 17). Upper clonograph: T cell receptor (TCR) in Tregs. Lower clonograph;
neutrop
neu
TC in Tcons. *Vβ9-Treg; p = 0.0031, **Vβ17-Treg; p = 0.0234, ***Vβ20-Treg; p = 0.0218,
TCR
****Vβ21.3-Treg;
p = 0.0262, *****Vβ17-Tcon; p = 0.0325 (Wilcoxon rank-sum test).
*** β
****V
Figure
4. The increased/decreased usage of T cell receptor (TCR)-Vβ families in CD4+CD25− T cells and
Fig
CD4+CD
CD
CD25+CD127low T cells of patients with autoimmune neutropenia (AIN) and control subjects
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Accepted Article
Means and
a standard deviations (SDs) of TCR-Vβ family usage of conventional T cells (CD4+CD25− T cells) and
regulatory T cells (CD4+CD25+CD127low T cells) were calculated in the control group and used as normal
regulato
reg
val
values.
Heat map classifies the degrees of each usage of TCR-Vβ families using five colors; within normal
range:
an −
−2 SD – +2 SD (white), > +2 SD (pink), > +3 SD (red), < −2 SD (pale green), < −3 SD (green) (a). The
increase
inc
increased and decreased numbers of CD4+CD25− T cell and CD4+CD25+CD127low T cells per person were
compare
com
compared. Data are represented as box-plots (b). *p = 0.0022, **p = 0.0001 (Wilcoxon rank-sum test). n.s; not
sig
significa
significant.
Fig
Figure
5. The different usage of T cell receptor (TCR)-Vβ families in individual conventional T cells
(Tcons) and regulatory T cells (Tregs)
Eac lin
Each
line presents individual usage of TCR-Vβ families in Tcon and Treg. *p < 0.05, **p < 0.01, ***p < 0.001,
****p
* < 0.0001 (Wilcoxon signed-rank test)
Figure 6. Preferential T cell subsets with higher usage of T cell receptor (TCR) Vβ families
Usa oof TCR Vβ families was compared between conventional and regulatory T cells, and the T cell subsets
Usage
wit hig
with
higher usage of TCR Vβ families were defined as preferential T cell subsets. Red color or pink color
indicate
ind
indicates a preferential subset, while blue color or light blue color indicates a decreased subset. White color
indicate
indicates no preference between conventional and regulatory T cells. Con T, conventional T cell; Reg T,
regulatory T cell. Wilcoxon signed-rank test: pink-light blue, p < 0.05; red-blue, p < 0.001.
regulato
reg
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cei_13559_f1.pdf
Accepted
eep ed Article
ept
icle
Figure1.
(a)
(b)
Total Tregs
CD4+ T cell
**
n.s
***
CD25
SSC
SC
CD25
Lymphcyte
gating
FSC
Foxp3
CD4
CD45RA
CD45RA
CD4+ T cell
III II
Foxp3
CD4
Treg subpopulations
I: Resting Tregs
II: Activated Tregs
III: Non suppressive Tregs
(c)
;ĚͿ
Lymphcyte
gating
CD127
SSC
SSC
CD4+ T cell
CD4
FSC
CD25
CD4+ T cell
Vȕ 7.1
2.50%
PE
Vȕ 5.3
0.75%
Vȕ3
9.06%
FITC
CD4+CD25- T cell
(conventional T cell)
Vȕ 5.3
0.72%
CD4+CD25+CD127low T cell
(regulatory T cell)
Vȕ 5.3
1.08%
Vȕ 7.1
1.70%
PE
PE
Vȕ 7.1
2.50%
Vȕ 3
7.35%
Vȕ3
9.32%
FITC
FITC
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cei_13559_f2.pdf
Accepted Article
Figure2
9ȕ *
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9ȕ
**
cei_13559_f3.pdf
Accepted Article
Figure3
Vȕ9
Vȕ17
**
Vȕ20
***
Vȕ21.3
****
Vȕ17
*****
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cei_13559_f4.pdf
AIN group
AI
up
Control grou
group
Cnt 1
Cnt 2
Cnt 2
Cnt 3
Cnt 3
Cnt 4
Cnt 4
Cnt 5
Cnt 5
Cnt 6
Cnt 6
Cnt 7
Cnt 7
Cnt 8
Cnt 8
Cnt 9
Cnt 9
Cnt 10
Cnt 10
Cnt 11
Cnt 11
Cnt 12
Cnt 12
Cnt 13
Cnt 13
Cnt 14
Cnt 14
Cnt 15
Cnt 15
Cnt 16
Cnt 16
Cnt 17
Cnt 17
Cnt 18
Cnt 18
Cnt 19
Cnt 19
Cnt 20
Cnt 20
Cnt 21
Cnt 21
Cnt 22
Cnt 22
AIN 1
AIN 1
AIN 2
AIN 2
AIN 3
AIN 3
AIN 4
AIN 4
AIN 5
AIN 5
AIN 6
AIN 6
AIN 7
AIN 7
AIN 8
AIN 8
AIN 9
AIN 9
AIN 10
AIN 10
AIN 11
AIN 11
AIN 12
AIN 12
AIN 13
AIN 13
AIN 14
AIN 14
AIN 15
AIN 15
AIN 16
AIN 16
Regulatory T cells
( CD4+CD25+CD127low T cells )
<-3SD <-2SD
-2SD ~ +2SD
Heat map
**
n.s
n.s
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>2SD >3SD
23
22
20
AIN 17
Conventional T cells
( CD4+CD25- T cells )
21.3
18
17
16
14
13.6
13.2
12
13.1
11
7.1
5.3
5.2
5.1
23
22
20
Cnt 1
AIN 17
(b)
21.3
18
17
16
14
TCR-Vȕ family
13.6
12
13.2
13.1
11
7.1
5.3
5.2
5.1
TCR-Vȕ family
7.2
(a
(a)
7.2
Accepted
pted Arti
Article
Figure4
Figure5
0.4
0.4
0.6
0.2
0.5
0.2
Treg
Treg
Tcon
Tcon
Treg
Treg
Tcon
Tcon
Treg
AIN
**
****
1Ϭ͘ϱ
0.5ϭ
0Ϭ
0Ϭ
1ϲ
Treg
Treg
Treg
Tcon
Treg
Tcon
Treg
Tcon
9ȕ
9ȕ
Cnt
AIN
Ϭ͘ϵ
0.7
0.6
Ϭ͘ϳ
0.5ϯ
Ϭ͘ϱ
0.4
0.3
Ϭ͘ϰ
0.2
0.1
Ϭ͘Ϯ
Ϭ͘ϴ
Ϭ͘ϲ
Ϭ͘ϯ
Ϭ͘ϭ
0Ϭ
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0.9
0.8
Treg
ϭ͘ϱ
Treg
Cnt
Treg
6ϯ͘ϱ
5ϯ
Tcon
ϭ͘Ϯ
Tcon
9ȕ
Cnt
AIN
Tcon
0Ϭ
1.5
Tcon
Treg
Ϭ͘Ϯ
Treg
Tcon
0.2
Ϭ͘ϱ
Ϯ͘ϱ
Treg
Tcon
Tcon
Treg
Ϭ͘ϰ
Treg
Treg
Treg
Tcon
ϭ͘ϱ
0.4
2.5
0Ϭ
Treg
Treg
Ϭ͘ϲ
1.5
Ϭ͘ϱ
0.5
Ϯ͘ϱ
1 Ϭ͘ϱ
ϯ0
Tcon
Ϭ͘ϴ
0.6
Treg
3.5ϲ
0.8
Ϯ͘ϱ
2ϭ͘ϱ
3ϱ
ϭ͘ϱ
0.5
ϯ͘ϱ
3.5
**
9ȕ
AIN
****
Tcon
45
4.5
Cnt
ϭ͘ϰ
2.5Ϯ
Tcon
ϱ͘ϱ
1.5
ϰ͘ϱ
6 ϯ͘ϱ
5ϯ
2.5
5.5
Tcon
Treg
Tcon
9ȕ
Cnt
AIN
**
ϯ͘ϱ
Treg
1.5
9ȕ
Cnt
AIN
1.2
Tcon
3ϯ
1.4
ϰ͘ϱ
3.5
Ϯ͘ϱ
Tcon
2.5
9ȕ
Cnt
AIN
Tcon
3ϱ
4ϰ
***
****
4.5ϯ͘ϱ
Treg
**
0Ϭ
3.5
Treg
Tcon
Tcon
Treg
Tcon
Treg
**
9ȕ
Cnt
AIN
3.5ϲ
0Ϭ
ϲ͘ϱ
Treg
9ȕ
Cnt
AIN
0Ϭ
Treg
Tcon
***
Tcon
00
0.5ϭ
Treg
6.5
9ȕ
Cnt
AIN
0.6
0 0
1ϭ
Tcon
Treg
Tcon
Tco
0Ϭ
1Ϭ͘ϱ
Treg
0.5
0.8
0.6
Ϭ0
0.8
0.2
2Ϯ
Tcon
0.21
ϭ͘ϱ
1 1
Treg
1.5
1.2
Ϭ͘ϱ
1.27
9ȕ
Cnt
AIN
0.4
Tcon
0.2
0.4
5ϱ
Ϯ͘ϱ
0.4
16
****
Tcon
9ȕ
AIN
Treg
2.5
7 6
Cnt
6ϲ
5ϯ
6 5
Treg
***
****
00
Tcon
****
10
1.2
Ϭ͘ϰ
Ϭ͘Ϯ
0.2
9ȕ
Cnt
AIN
****
Ϭ͘ϴ
Ϭ͘ϲ
0.4
Tcon
****
6ϯ͘ϱ
3ϭ
ϭ͘ϱ
Tcon
Treg
9ȕ
Cnt
AIN
AIN
ϭ͘ϰ
ϭ͘Ϯ
0.8
00
Tcon
Treg
Tcon
35
3.5
Ϯ͘ϱ
1.4
Treg
Tcon
1.2
0.6
0.8
1.5
0.6
9ȕ
ϭ͘ϴ
ϭ͘ϲ
Cnt
6Ϯ
1.6
1.2
2.5
9ȕ
Cnt
AIN
1.8
1.2
0.8
.8
01
ϯ͘ϱ
**
1.4
0.6
1.5
Tcon
3.5
1.6
1.5
Treg
***
9ȕ
Cnt
AIN
0.8
0.6
0.4
0.2
Treg
00
0.8
Treg
10
24
Treg
9ȕ
AIN
1.6
2.5
***
1.4
38
2.5
****
1.4
1.2
61
1.5
Treg
Treg
Cnt
Tcon
Tcon
Treg
1.5
10
86
1.6
10
10
Tcon
**** ****
3.5
10
Tcon
12
9ȕ
Cnt
AIN
9ȕ
AIN
Cnt
12
3.5
Tcon
14
10
12
3.5
9ȕ
AIN
Cnt
**
Tcon
2.5
*****
14
9ȕ
AIN
****
12
16
10
2.5
1.6
Cnt
Treg
3.5
1.4
9ȕ
AIN
16
4.5
Cnt
Tcon
4.5
AIN
Tcon
9ȕ
Accepted
Acc
Ac
cc p
cc
ptte
pte
ted Ar
Article
rticle
ticle
cle
cl
Cnt
cei_13559_f5.pdf
Accepted Article
cei_13559_f6.pdf
Figure 6
TCR Vb
5.1
5.2
5.3
7.1
7.2
11
12
13.1
13.2
13.6
14
16
17
18
20
21.3
22
23
Control
Con T
Reg T
AIN
Con T
Reg T
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...