PMC5430328_01
PMC5430328_02
(A) Frequency of peripheral blood CD4+CD25+Foxp3+ regulatory T-cells (Tregs) in I-cSLE, active cSLE (A-cSLE) patient, and healthy control (HC). Frequency of Tregs (CD4+CD25+Foxp3+) under basal conditions in the HC, inactive childhood-onset systemic lupus erythematous (cSLE) patient, and A-cSLE patient. (B) Frequency of CD25hiFoxp3hi peripheral blood Tregs in I-cSLE patient, A-cSLE patient, and HC. Frequencies of CD4+CD25hi Foxp3hi under basal conditions in the HC, inactive cSLE patient, and A-cSLE patient.
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Both the inactive (I-cSLE) and A-cSLE patients possessed high frequencies of CD4+CD25+Foxp3+ Tregs (5-12%) compared to the HC, who had 3%Tregs (Figure 2A). Tregs percentages: 4   12   12% (Figure 2A). The active lupus patient also had a higher percentage of CD25hiFoxp3hi Tregs at time points 2 and 3 (after initiation of therapy), but not at time point 1 compared to the HC (Figure 2B).
Ratio of regulatory T-cells to Teff in active cSLE (A-cSLE) patient over time points compared to I-cSLE and healthy control (HC). Samples from A-cSLE patient were collected at three time points (1, 2, and 3). Samples from HC and I-cSLE patient were collected once, represented as time point 1.
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In the HC, this ratio was 0.36, and in the I-cSLE patient, the ratio was 0.96 (Figure 3).
Distribution of CD4+ T-cell subsets in I-cSLE patient, active cSLE patient, and healthy control under basal and post-stimulatory conditions. Peripheral blood mononuclear cells were stimulated (S) with super-antigen staphylococcal enterotoxin B for 24 h and then compared with their corresponding samples under basal conditions (B) for frequencies of different CD4+ T-cell subsets.
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Tnaive/mem cells at time point 1 under basal (non-stimulated) conditions between the HC and lupus patients (Figure 4). The HC had 7.93% Teff cells, I-cSLE patient had 5.12% Teff cells, and A-cSLE patient had 6.76, 9.34, and 7.67% Teff cells at time point 1, 2, and 3, respectively. The HC had 88.8% Tnaive/mem cells, I-cSLE patient had 87.6% Tnaive/mem cells, and A-cSLE patient had 86, 76.5, and 75.8% Tnaive/mem cells at time point 1, 2, and 3, respectively (Figure 4). I-cSLE patient compared to the HC and the A-cSLE patient with a 9.10-fold increase in the percentages of CD4+ Teff cells after stimulation with SEB (Figure 4). Interestingly, the fold change of CD4+ Teff cells in the A-cSLE patient gradually increased after induction of therapy from time points 1 to 3: 1.43-fold   3.87-fold   5.51 (Figure 4). I-cSLE patient showed 2-fold reduction in this subset (Figure 4). In the A-cSLE patient, this reduction in the Tnaive/mem subset became more pronounced from time points 1 to 3 from 1.02-fold   1.6-fold   2.04-fold as her disease activity gradually improved with SLEDAI scores decreasing from 14   10   8 (Figure 4).
Surface expression of PD-1 on Teff, regulatory T-cells (Tregs), and Tnaive/mem cells under basal conditions. Frequencies of PD-1 expressing Teff, Tregs, and Tnaive/mem cells under basal (non-stimulatory) conditions in I-cSLE patient, active cSLE patient, and healthy control.
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T-cell subsets including Teff, Tregs, and Tnaive/mem cells to assess for differences between active and inactive lupus patients as PD-1 has been shown to play a role in T-cell exhaustion. PD-1 was expressed at much higher percentages on CD4+ Teff, Tregs, and Tnaive/mem cells at all three time points in the A-cSLE patient compared to the I-cSLE patient and HC (Figure 5). The active patient had PD-1 expressing Teff cells that ranged from 4.4 to 8.37%, the inactive patient had 2.9% PD-1 expressing Teff cells, and the HC had 1%.