Characteristics of SLE patients
Given recent studies showing the imbalances in the effector and regulatory Tfh cells compartment in SLE patients [3, 4], and in light of our finding that low-dose IL-2 treatment significantly influences Tfh subtypes, we recruited a small cohort of healthy controls (HC) (n=23, Table 1) for comparative analysis.
The demographic and clinical manifestations of these patients were shown in Table 1. There was no significant difference between patients and HCs regarding age or gender. 98% of the SLE patients were positive in anti-dsDNA tests, 42% had renal involvement and 48% had skin manifestations.
Imbalanced Tfr/Tfh in SLE
As shown in Table 2, regulatory T cells including Treg, CXCR5+PD-1lowTreg, CXCR5+PD-1highTreg cells were significantly reduced in SLE patients than those in HC (P=0.087, P=0.033, P<0.001, P<0.001, respectively). In contrast, the effector Tfh cells were significantly increased in SLE patients than in HC (p=0.081). Besides, Tfr subsets: Tfh subsets ratios in SLE were dramatically decreased, including CXCR5+PD-1lowTreg/Tfh (P=0.043), CXCR5+PD-1highTreg/Tfh (p<0.001), CXCR5+PD-1lowTreg/Tfh17 (P=0.052) and CXCR5+PD-1highTreg/Tfh17 (P<0.001).
High Tfh associated with decreased serum IL-2
As show in Figure 2, an expanded inflammatory Tfh cell compartment was correlated with higher serum IL-17 levels (r=0.328, P=0.021) and increased frequency of Tfh17 correlated with lower serological IL-2 (r=-0.295, P=0.04) (Figure 2). There was a reduction of CXCR5/PD-1 double positive subset (CXCR5+PD1highTreg), which was related with IL-10 elevation (r=0.243, P=0.093, Figure 3).
Table 2. Difference of CD4 T subsets between HC and SLE, and between active and remission group.
|
Variables
|
HC
(n=23)
|
SLE
|
P,
HC VS. Active
|
P, Remission VS. Active
|
Active (n=60)
(Before therapy)
|
Remission (n=59)
(After therapy)
|
Proportion (percentage in lymphocyte, %)
|
Treg
|
1.22 (1, 1.47)
|
0.95 (0.65, 1.53)
|
1.6 (0.97, 2.49)
|
0.087
|
0.001
|
CXCR5+PD-1lowTreg
|
0.09 (0.04, 0.14)
|
0.06 (0.03, 0.1)
|
0.13 (0.06, 0.25)
|
0.033
|
<0.001
|
CXCR5+PD-1highTreg
|
0.09 (0.07, 0.13)
|
0.02 (0.01, 0.04)
|
0.11 (0.06, 0.19)
|
<0.001
|
<0.001
|
Tfh
|
0.28 (0.17, 0.43)
|
0.39 (0.24, 0.56)
|
0.19 (0.09, 0.37)
|
0.081
|
0.002
|
Tfh1
|
0.61 (0.38, 0.99)
|
0.2 (0.08, 0.36)
|
0.17 (0.09, 0.28)
|
<0.001
|
0.42
|
Tfh2
|
1.06 (0.58, 1.5)
|
0.42 (0.21, 0.6)
|
0.36 (0.19, 0.68)
|
<0.001
|
0.367
|
Tfh17
|
0.45 (0.15, 0.65)
|
0.47 (0.27, 0.87)
|
0.44 (0.2, 0.83)
|
0.345
|
0.851
|
Absolute number (cells/L)
|
Treg
|
25.51 (19.98, 30.78)
|
11.94 (6, 20.81)
|
20.51 (9.17, 29.06)
|
0.035
|
0.014
|
CXCR5+PD-1lowTreg
|
2.98 (1.71, 3.1)
|
0.67 (0.24, 1.21)
|
1.33 (0.8, 3.54)
|
0.037
|
0.005
|
CXCR5+PD-1highTreg
|
2.99 (1.18, 3.57)
|
0.24 (0.06, 0.56)
|
1.5 (0.71, 2.27)
|
0.001
|
<0.001
|
Tfh
|
7.29 (4.34, 8.92)
|
4.6 (2.58, 10.28)
|
2.84 (0.81, 6.04)
|
0.625
|
0.008
|
Tfh1
|
14.26 (12.37, 18.22)
|
2.11 (0.95, 5.47)
|
1.92 (0.68, 4.28)
|
0.002
|
0.427
|
Tfh2
|
19.31 (14.66, 24.22)
|
4.43 (2.11, 11.2)
|
4.04 (1.73, 11.88)
|
0.002
|
0.751
|
Tfh17
|
8.82 (7.25, 12.22)
|
5.39 (2.33, 14.38)
|
4.67 (2.47, 10.36)
|
0.642
|
0.664
|
Ratios
|
|
|
|
|
|
Treg/Tfh
|
3.69 (3.04, 8.49)
|
2.49 (1.89, 4.5)
|
8.93 (4.04, 18.29)
|
0.002
|
<0.001
|
CXCR5+PD-1lowTreg/Tfh
|
0.3 (0.21, 0.57)
|
0.16 (0.08, 0.31)
|
0.61 (0.4, 1.32)
|
0.002
|
<0.001
|
CXCR5+PD-1highTreg/Tfh
|
0.46 (0.21, 0.69)
|
0.05 (0.01, 0.1)
|
0.54 (0.24, 1.3)
|
<0.001
|
<0.001
|
Treg/Tfh17
|
3.87 (2.27, 5.71)
|
2.29 (1.23, 4.21)
|
3.89 (2.01, 6.62)
|
0.047
|
<0.001
|
CXCR5+PD-1lowTreg/Tfh17
|
0.25 (0.17, 0.34)
|
0.11 (0.05, 0.22)
|
0.28 (0.18, 0.52)
|
0.002
|
<0.001
|
CXCR5+PD-1highTreg/Tfh17
|
0.3 (0.14, 0.64)
|
0.03 (0.01, 0.09)
|
0.23 (0.12, 0.45)
|
<0.001
|
<0.001
|
SLE, systemic lupus erythematosus. HC, healthy controls. Data are median (IQR).
|
Imbalanced Tfh and Tfr cell association with disease activity in SLE
Treg cells were decreased and associated with elevated ESR (r=-0.382, P<0.01) and Safety of Estrogens in Lupus Erythematosus National Assessment version of the SLE Disease Activity Index (SLEDAI) (r=-0.245, P=0.089, Figure 2). At the same time, the decrease of CXCR5+PD-1lowTreg was associated with increased ESR and anti-dsDNA antibodies production (Figure 2, 3). In contrast, an increased inflammatory Tfh cell compartment was found and was correlated with elevated SLEDAI, titer of anti-AnuA, anti-dsDNA antibodies, serum IL-17 and decreased C3 (Figure 2, 3).
Upon further analysis of the correlation between regulatory and effector subsets, we found decreased Treg/Tfh ratio in severe patients with higher SLEDAI score, higher titers of anti-AnuA and anti-dsDNA antibodies (Figure 3). In addition, there was a reduced CXCR5+PD1lowTreg/Tfh in this group of severe patient.
Figure 3 showed that the frequency of Tfh1 and Tfh2 was positively correlated with the number of total B cells and switched memory B (CD19+IgD-CD27+) cells. CXCR5+PD-1lowTreg/Tfh17 was negatively correlated with switched memory B cells and plasma B cells (r=-0.341, P=0.027, Figure 3). And decreased CXCR5+PD-1lowTreg/Tfh and CXCR5+PD-1lowTreg/Tfh17 were both associated with increased serum level of IgM.
Low-dose IL-2 therapy increased Tfr/Tfh ratio in SLE patients
In this RCT of low-dose IL-2 therapy in SLE, low-dose IL-2 significantly increased Tregs [10]. With effective treatment, British Isles Lupus Assessment Group (BILAG), SLEDAI, SLE Responder Index-4 (SRI-4), physician’s global assessment (PGA), myositis, fever, alopecia, vasculitis, arthritis, oral ulcer and rash were all improved at week 12 (Figure 4) [10]. After 3 cycles of low-dose IL-2 therapy, the frequency of CXCR5+PD-1lowTreg cells and CXCR5+PD-1highTreg cells in lymphocyte was significantly increased at week 12 compared to placebo control (0.06 (0.03, 0.1) vs. 0.13 (0.06, 0.25), p<0.001 and 0.02 (0.01, 0.04) vs. 0.11 (0.06, 0.19), P<0.001 respectively) (Table 2, Figure 4). Similarly, the absolute number of these Treg cells (CXCR5+PD-1highTreg and CXCR5+PD-1lowTreg) were significantly increased after the treatment of low-dose IL-2 (0.67 (0.24, 1.21) vs. 1.33 (0.8, 3.54), P=0.005 and 0.24 (0.06, 0.56) vs. 1.5 (0.71, 2.27), P<0.001 respectively) (Table 2, Figure 4). Besides, compared to baseline, Tfr subsets: Tfh subsets ratios in SLE were dramatically increased, including CXCR5+PD-1lowTreg/Tfh (P<0.001), CXCR5+PD-1highTreg/Tfh (p<0.001), CXCR5+PD-1lowTreg/Tfh17 (P<0.001) and CXCR5+PD-1highTreg/Tfh17 (P<0.001).