Supplementary document for Modified rigorous coupled-wave analysis for multi-layer deformable gratings with arbitrary profiles and materials - 6111619.pdf

Supplemental Document final versio

Effect of alemtuzumab concentration on survival of NK cells from the liver and peripheral blood.

<p>Cell survival was evaluated using Annexin V and propidium iodide staining. Mononuclear cells were incubated alone or in the presence of alemtuzumab (100, 10, 1, or 0.1 μg/mL) for different time periods (1 and 4 h). The surviving cells were negative for both Annexin V and propidium iodide. (A) The survival rate of LMNCs after co-incubation with alemtuzumab was significantly higher than that of PBMCs for each concentration. The numbers present the proportion of each subset. The dot plots are representative of 7 independent experiments (alemtuzumab; 100 μg/mL, 4-h culture). The bar graphs show the mean survival rate ± SD of LMNCs and PBMCs after 4-h treatment at each concentration (n = 7, *p < 0.05 by Student’s <i>t</i>-test). (B) The survival rate of liver NK cells was significantly higher than that of liver-derived non-NK cells (n = 4, *p < 0.05 by Student’s <i>t</i>-test). (C) CD56^bright NK cells survived after co-incubation with alemtuzumab, compared to CD56^dim NK cells (n = 4, *p < 0.05 by Student’s <i>t</i>-test).</p

The liver contained a high percentage of CD52⁻ NK cells.

<p>CD52⁻ and CD52⁺ NK cells were defined as follows: Cells were gated on the CD3⁻CD56⁺ NK cell, CD3⁻CD56^bright, or CD3⁻CD56^dim populations within singlet and lymphocyte gates. A CD52⁻ gate was set using the isotype control. Data are representative of 7 separate experiments. (A) CD52 expression on LMNCs and PBMCs. CD52 expression levels on LMNCs and PBMCs were evaluated by FCM. LMNCs contain a significantly larger proportion of CD52⁻ cells when compared with PBMCs. The bar graph shows the mean ± SD of CD52⁻ lymphocytes in the liver and peripheral blood (n = 7, *p < 0.05 by Student’s <i>t</i>-test) (B) CD52 expression on NK cells in the liver and peripheral blood. CD52 expression on NK cells from the liver was significantly lower than that on NK cells from the peripheral blood. The bar graph shows the mean ± SD of CD52⁻ NK cells derived from LMNCs and PBMCs (n = 7, *p < 0.05 by Student’s <i>t</i>-test) (C) CD52 expression on liver CD56^bright and liver CD56^dim NK cells. CD52 expression levels of each population were calculated by FCM. About 90% of CD56^bright liver NK cells (10.2% ± 5.7) did not express CD52. CD52 expression on CD56^dim NK cells was significantly higher when compared with that on CD56^bright liver NK cells. The bar graph shows the mean ± SD of CD52⁻ cells in each NK cell population (n = 7, *p < 0.05 by Student’s <i>t</i>-test).</p

CD52⁺ and CD52⁻ NK cells from the liver had different FACS profiles.

<p>(A) Comparison of CD52⁺ NK cells and CD52⁻ NK cells in the liver. The representative histograms of 7 independent experiments are shown for CD52⁺ NK cells (solid line) and CD52⁻ NK cells (dotted line). The gray solid line shows the isotype control. (B) Dot shows the percentage of each surface marker on CD52^- and CD52⁺ cells. The solid line indicates mean value in each population and two points connected by dotted line indicate these cells are from same donor. (*p < 0.05 by Student’s paired <i>t</i>-test). (n = 4 or 7, *p < 0.05).</p