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. 2020 Nov 11;11:592553. doi: 10.3389/fimmu.2020.592553

Figure 6.

Figure 6

The effect of siplizumab (A, B), Alemtuzumab (C, D) and rabbit anti-thymocyte globulin (rATG; E, F) on CD69 and HLA-DR expression on T cells in allogeneic mixed lymphocyte reaction (MLR). Expression of CD69 and HLA-DR were analyzed at baseline (day 0) and on days 1, 2, 4, and 7. Data is displayed as the mean of all data points (N = 9 donor pairs) ± SD. T cells were identified as CD3+ CD56−. Data were analyzed using two-way ANOVA followed by Dunnett’s multiple comparison test with untreated controls (no antibody) serving as the comparison data set (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (A) Siplizumab significantly decreased CD69+ T cells on day 2 at 0.1–10 µg/ml (p ≤ 0.0490) and on day 4 at 10 µg/ml siplizumab (p = 0.0351). (B) HLA-DR expression on T cells was significantly inhibited on days 2, 4, and 7 at 0.1 µg/ml (p = 0.0104; p = 0.0373; p = 0.0008), 1 µg/ml (p = 0.0059; p = 0.0189; p = 0.0005), and 10 µg/ml (p = 0.0021; p = 0.0190; p = 0.0005) siplizumab. (C) Alemtuzumab significantly increased CD69+ T cells after 1 day of allogeneic MLR at 0.01–10 µg/ml (p ≤ 0.0091). On days 2, 4, and 7, no dose-dependent change in CD69 expression was observed. (D) Alemtuzumab significantly increased HLA-DR+ T cells on day 1 at 0.01–10 µg/ml (p ≤ 0.0344), on day 2 at 0.1 µg/ml (p = 0.0167) and 1 µg/ml (p = 0.0157), as well as on day 4 at 1 µg/ml (p = 0.0261) and 10 µg/ml (p = 0.0010). No significant differences were detected on day 7. (E) A significant increase in CD69+ T cells was detected after 1, 2, and 4 days of allogeneic MLR in samples treated with 0.001 µg/ml (p ≤ 0.0467), 0.01 µg/ml (p ≤ 0.0024), 0.1 µg/ml (p ≤ 0.0017), 1 µg/ml (p ≤ 0.0007), and 10 µg/ml rATG (p < 0.0001). After 7 days, rATG induced a significant increase in CD69 expression on T cells at 0.01–10 µg/ml (p < 0.0001). (F) rATG induced a modest but statistically significant increase in HLA-DR+ T cells after 1 day of allogeneic MLR at 0.01–10 µg/ml (p ≤ 0.0056) as well as after 2 days at 0.01–1 µg/ml (p < 0.0159). In contrast, a decrease in HLA-DR expression in rATG-treated samples was noted at 0.01–10 µg/ml; however, only 0.01 µg/ml (p = 0.0170) and 10 µg/ml (p = 0.0048) were significant. (G) Representative histograms showing CD69 expression on T cells after 1 day of MLR (upper panel) and HLA-DR expression after 7 days of MLR (lower panel) in samples treated with 10 µg/ml siplizumab or 10 µg/ml rATG.