Increased percentages of CD134+CD4+ T-cells in SLE patients with LN
First, we studied the expression of CD134+CD4+ T-cells in unstimulated peripheral blood cells. The percentages of CD134+CD4+ T-cells were significantly increased in SLE patients as compared to healthy controls (21.4 ± 10.4% vs. 13.7 ± 5.8%; p = 0.002). There was no significant difference between the percentages of CD134+CD4+ T-cells in active SLE patients as compared to inactive SLE patients (24.8 ± 12.2% vs. 20.6 ± 10.1%; p = 0.42). The percentages of CD134+CD4+ T-cells were significantly increased in active SLE patients as compared to healthy controls (24.8 ± 12.2% vs. 13.7 ± 5.8%; p = 0.02) and between inactive SLE patients versus healthy controls (20.6 ± 10.1 vs. 13.7 ± 5.8%; p = 0.006). Next, we compared SLE patients with LN to SLE patients without LN. The percentages of CD134+CD4+ T-cells were significantly increased in SLE patients with LN as compared to SLE patients without LN and HC, respectively (25.7 ± 10.5% vs. 19.2 ± 9.9%; p = 0.046; 25.7 ± 10.5% vs. 13.7 ± 5.8%; p = 0.0007).
Increased percentages of PD1+CD4+ T-cells in SLE patients with LN
First, we studied the expression of PD1+CD4+ T-cells in unstimulated peripheral blood cells. The percentages of PD1+CD4+ T-cells were not significantly increased in SLE patients as compared to healthy controls (35.9 ± 15.8% vs. 29.2 ± 5.8%, p = 0.06). There was no significant difference between active patients SLE patients as compared to inactive SLE patients (38.2 ± 23.3% vs. 35.4 ± 14.1%; p > 0.99) and to HC, respectively (38.2 ± 23.3% vs. 29.2 ± 5.8%; p = 0.73). The percentages of PD1+CD4+ T-cells were significantly increased in inactive SLE patients as compared to HC (35.4 ± 14.1% vs. 29.2 ± 5.8%; p = 0.04).
The percentages of PD1+CD4+ T-cells were significantly increased in SLE patients with LN as compared to HC (41.0 ± 13.6% vs. 29.2 ± 5.8%; p = 0.004). There was no significant difference between SLE patients with LN as compared to SLE patients without LN (41.0 ± 13.6% vs. 33.4 ± 16.4%; p = 0.25). There was no significant difference between SLE patients without LN and HC (33.4 ± 16.4% vs. 29.2 ± 5.8%; p = 0.4).
Decreased IFN-γ and IL-22 secretion in stimulated CD134+CD4+ T-cells
Next, we studied the cytokine production of IFN-γ, IL-21 and IL-22 in CD134+CD4+ T-cells of SLE patients and HC after in vitro stimulation. The percentages of IFN-γ-producing CD134+CD4+ T-cells were significantly decreased in SLE patients as compared to HC (18.5 ± 11.5% vs. 29.2 ± 19.4%; p = 0.02, Fig. 1). There was no significant difference between active and inactive SLE patients (16.8 ± 10.7% vs. 18.8 ± 11.8%; p = 0.74) and between active SLE patients and HC (16.8 ± 10.7% vs. 29.2 ± 19.4%; p = 0.08). The percentages of IFN-γ-producing CD134+CD4+ T-cells in inactive patients were significantly decreased as compared to HC (18.8 ± 11.8% vs. 29.2 ± 19.4%; p = 0.04). There were no intraindividual differences in SLE patients between two outpatient visits (Fig. 3).
The percentages of IFN-γ-producing CD134+CD4+ T-cells were significantly increased in SLE patients with lupus nephritis as compared to patients without lupus nephritis (25.4 ± 11.4% vs. 15.0 ± 10.1%; p = 0.007, Fig. 4). The percentages of IFN-γ-producing CD134+CD4+ T-cells were also significantly increased in HC as compared to patients without lupus nephritis (29.2 ± 19.4% vs. 15.0 ± 10.1%; p = 0.001). SLE patients with lupus nephritis had no differences in the percentages of IFN-γ-producing CD134+CD4+ T-cells as compared to HC (25.4 ± 11.4% vs. 29.2 ± 19.4%; p = 0.99).
The percentages of IL-21-producing CD134+CD4+ T-cells were not significantly different between SLE patients and HC (11.9 ± 11.2% vs. 16.6 ± 19.0%; p = 0.73). There was also no difference between active and inactive SLE patients (12.5 ± 9.1% vs. 11.7 ± 11.7%; p = 0.63), active SLE patients and HC (12.5 ± 9.1% vs. 16.6 ± 19.0%; p = 0.90), and inactive SLE patients and HC (11.7 ± 11.7% vs. 16.6 ± 19.0%; p = 0.65). There was also no difference between the percentages of IL-21-producing CD134+CD4+ T-cells in SLE patients with lupus nephritis as compared to patients without lupus nephritis (12.5 ± 12.7% vs. 11.6 ± 10.6%; p = 0.89). Accordingly, no differences were found comparing patients with lupus nephritis versus HC, respectively (12.5 ± 12.7% vs. 16.6 ± 19.0%; p = 0.75).
The percentages of IL-22-producing CD134+CD4+ T-cells were significantly decreased in SLE patients as compared to HC (10.5 ± 9.0% vs. 19.0 ± 11.8%; p = 0.01). There was no significant difference between the percentages of IL-22-producing CD134+CD4+ T-cells in active SLE patients as compared to inactive SLE patients (8.0 ± 8.4% vs. 10.9 ± 9.2%; p = 0.63) and active SLE patients as compared to HC (8.0 ± 8.4% vs. 19.0 ± 11.8%; p = 0.06). There was a significant difference regarding the percentages of IL-22-producing CD134+CD4+ T-cells in inactive SLE patients as compared HC (10.9 ± 9.2% vs. 19.0 ± 11.8%; p = 0.02).
There was no significant difference comparing the percentages of IL-22-producing CD134+CD4+ T-cells in SLE patients with lupus nephritis versus patients without lupus nephritis (13.5 ± 9.7% vs. 9.0 ± 8.4%; p = 0.19) and HC, respectively. (13.5 ± 9.7% vs. 19.0 ± 11.8%; p = 0.13). There was also no significant difference with respect to the percentages of IL-22-producing CD134+CD4+ T-cells in SLE patients without lupus nephritis as compared to HC (9.0 ± 8.4% vs. 19.0 ± 11.8%; p = 0.008).
Decreased IFN-γ secretion of PD1+CD4+ T-cells in SLE
Following the analysis of PD-1 surface expression on CD4+ T-cells, we studied the cytokine expression of IFN-γ, IL-21 and IL-22 in PD-1+CD4+ T-cells of SLE patients and HC after in vitro stimulation. The percentages of IFN-γ-producing PD-1+ CD4+ T-cells were significantly decreased in SLE patients as compared to healthy controls (31.8 ± 15.6% vs. 41.7 ± 14.3%; p = 0.03, Fig. 2). The percentage of IFN-γ-producing PD-1+ CD4+ T-cells in SLE patients was stable between two outpatient visits (Fig. 3). The comparison of active versus inactive SLE patients and active SLE patients versus HC showed no significant difference regarding the percentages of IFN-γ-producing PD-1+CD4+ T-cells (38.0 ± 20.9% vs. 30.4 ± 14.3%; p = 0.40 and 38.0 ± 20.9% vs. 41.8 ± 14.3%; p = 0.75, respectively). Inactive SLE patients had significantly decreased percentages of IFN-γ-producing PD-1+CD4+ T-cells as compared to HC (30.4 ± 14.3% vs. 41.7 ± 14.3%; p = 0.02). There was no difference when comparing the percentages of IFN-γ-producing PD-1+CD4+ T-cells in SLE patients with LN versus SLE patients without LN (36.1 ± 14.0% vs. 29.6 ± 16.2%; p = 0.20); likewise, no difference was found between SLE patients with LN and HC (36.1 ± 14.0% vs. 41.8 ± 14.3%; p = 0.40). SLE patients without LN had significantly decreased percentages of IFN-γ-producing PD-1+CD4+ T-cells as compared to HC (29.6 ± 16.2% vs. 41.8 ± 14.3%; p = 0.02).
The percentages of IL-21-producing PD-1+ CD4+ T-cells were not significantly different in SLE patients as compared to healthy controls (12.6 ± 11.0% vs. 14.7 ± 16.2%; p = 0.91, Fig. 2). The percentages of IL-21-producing PD-1+CD4+ T-cells were also not significantly different in active SLE patients as compared to inactive SLE patients (14.5 ± 8.0% vs. 12.11 ± 11.6%; p = 0.30) and as compared to HC (14.5 ± 8.0% vs. 14.7 ± 16.2%; p = 0.53). There was also no difference in the production of IL-21 in PD-1+ CD4+ T-cells of SLE patients with LN as compared to SLE patients without LN (11.6 ± 10.1% vs. 13.0 ± 11.6%; p = 0.79). Similarly, there was no statistically significant difference comparing SLE patients with LN versus HC (11.6 ± 10.1% vs. 14.7 ± 16.2%; p = 0.98, Fig. 4).
The fraction of IL-22-producing PD-1+ CD4+ T-cells was not significantly different in SLE patients as compared to HC (6.4 ± 6.5% vs. 10.2 ± 11.6%; p = 0.26). The percentages of IL-22-producing PD-1+ CD4+ T-cells were also not significantly different comparing active and inactive patients (3.4 ± 2.6% vs. 7.1 ± 6.8%; p = 0.17) as well as active patients versus HC, respectively (3.4 ± 2.6% vs. 10.2 ± 11.6%; p = 0.10). There were no differences between all other groups analyzed.
Influence of immunosuppressive treatment
To analyze the cytokine-secreting capacity of CD134+ and PD-1+CD4+ T-cell subsets dependent on the immunosuppressive treatment, we stratified the patients in two groups. We compared patients who received MMF to patients with an immunosuppressive regimen without MMF. There was no significant difference between these groups regarding effector cytokine production (IFN-γ, IL-21 and IL-22) within CD134+ and PD-1+CD4+ T-cells (Fig. 5a). To investigate the role of prednisone, we correlated the daily dose of prednisone in mg/day with these T-cell subsets. There was no significant correlation found in these groups (Fig. 5b).