Figure 4.

The effect of siplizumab (A, B), Alemtuzumab (C, D), and rabbit anti-thymocyte globulin (rATG) (E, F) on naïve CD4 T cell (Tn) enrichment in allogeneic mixed lymphocyte reaction (MLR). T cell subpopulations 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. CD4 and CD8 T cells were identified as CD3⁺ CD56⁻ CD4⁺ and CD3⁺ CD56⁻ CD8⁺ lymphocytes, respectively. Tn were identified as CD45RA⁺ CCR7^high. 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 enriched CD4+ Tn at all tested concentrations after 1 day of allogeneic MLR (p ≤ 0.0342) as well as at 0.001–10 µg/ml after 2 (p ≤ 0.0077) and 4 (p ≤ 0.0187) days. After 7 days, statistically significant enrichment of CD4+ Tn was observed in samples treated with 0.1 µg/ml (p = 0.0423) and at 0.1–10 µg/ml (p < 0.0001) siplizumab. (B) Siplizumab enriched CD8+ Tn at all tested concentrations after 1 day (p ≤ 0.0398), at or above 0.001 µg/ml after 2 days (p ≤ 0.0039), at or above 0.01 µg/ml after 4 days (p ≤ 0.019) and at 0.1 µg/ml or higher after 7 days (p ≤ 0.0044) of allogeneic MLR. (C) Alemtuzumab increased CD4+ Tn at 0.0001–1 µg/ml on day 1 (p ≤ 0.0362), 0.01–10 µg/ml on day 2 (p ≤ 0.0255), and 0.01–0.1 µg/ml on day 4 (p ≤ 0.0005) of allogeneic MLR. No statistically significant enrichment of CD4 Tn by Alemtuzumab was detected on day 7. (D) No notable enrichment of Tn among CD8 T cells was detected in Alemtuzumab-treated samples. (E) rATG enriched CD4+ Tn at 0.001 µg/ml (p = 0.0411) and 0.1–10 µg/ml (p ≤ 0.0285) on day 1, at 0.01–10 µg/ml (p = 0.0411) on day 2 (p ≤ 0.0338), and at 0.01 µg/ml on day 4 (p = 0.0470) of allogeneic MLR. Moreover, 10 µg/ml rATG induced a significant depletion of CD4+ Tn on day 7 (p = 0.0273). (F) No clear dose-dependent enrichment or depletion of CD8+ Tn occurred in rATG-treated samples. (G) Representative dot plots illustrating differences in subpopulation distribution among CD4 and CD8 T cells in MLRs treated with no antibody, 10 µg/ml siplizumab, 10 µg/ml Alemtuzumab, and 10 µg/ml rATG. Tcm, central memory T cells, CD45RA⁻ CCR7^high; Tn, naïve T cells, CD45RA⁺ CCR7^high; Temra, CD45RA⁺ CCR7^low, effector memory CD45RA+ T cells; tem, Effector memory T cells, CD45RA⁻ CCR7^low.