Fig 2: Differences in lymphocytes counts of peripheral blood mononuclear cells (PBMCs) in peripheral blood in lung cancer patients and healthy controls. (a) Flow cytometry was performed to detect lymphocyte subsets in PBMCs and to analyze the proportions of T cells (CD3+CD56−), B cells (CD19+), natural killer T (NKT) cells (CD3+CD56+), and natural killer (NK) cells (CD3−CD56−) among CD45+ cells and the proportions of CD4+ T cells and CD8+ T cells among T cells. The results are presented using scatter plots. (b) Differences in T cells (CD3+CD56−), B cells (CD19+), NKT cells (CD3+CD56+), and NK cells (CD3−CD56−) among CD45+ cells and the proportions of CD4+ T cells and CD8+ T cells among T cells between lung cancer patients and healthy controls. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig 3: Flow cytometry analysis of peripheral immune cells in blood from normotensive control and pre-eclampsia (PE) women taken before delivery. (A) The relative proportion of T cells and natural killer (NK) cells. (B) Flow cytometry analysis of CD69+, CD11a+, CD8+, and CD4+ T cells. (C) Analysis of CD3–, CD56+, and CD16+ NK cells, divided into CD56 Bright (Gate A) and Dim NK cells (Gate B), and early activation CD69+.

Fig 4: Lymphocyte activation by choriocarcinoma cells. (A) PBMCs with HLA-C haplotype Cw03 and Cw07 were exposed to JAr (ERAP2+, HLA-C−), JEG-3 (ERAP2−, HLA-C+), or T3M-3 (ERAP2+, HLA-C+) cells, and they were then analyzed by flow cytometry for the detection of CD3 (all T cells), CD8 (T cells), CD56 (NK cells), and CD69 (activation). Percentages in the dot plots represent the active CD3+CD8+CD69+ T cells or CD3–CD56+CD69+ NK cells. (B) The histogram depicts the calculated percentage of subpopulations of T-cell activation (CD8+, CD69+) and NK-cell activation (CD56+, CD69+) in response to incubation with each cell preparation. Each experiment was repeated three times with three replicates in each experiment. There was significant activation of NK cells exposed to T3M-3 cells (one way ANOVA statistical analysis: * = P < 0.05), while T-cell activation was not significant compared to T cells exposed to other cell lines.

Fig 5: Lymphocytes attach and kill trophoblasts expressing ERAP2. (A) A transfected GFP-producing JEG-3 trophoblast cell can be seen with a T cell and NK cell attached. The T cell is labeled with APC/Fire 750 anti-CD3 (dark pink) and the NK cell is labeled with PE–CD56 (yellow) and BV421 anti-CD40L (purple). Cells transfected with the empty vector (GFP+ = green) are larger and healthier and do not have T or NK cells attached. (B) Dot plots from flow cytometry showed differences in the healthy populations between cells transfected with N392 or K392 ERAP2, or an empty vector and nontransfected cells when exposed to PBMCs. Live, rounded, and healthy cells appear on the top left plot along the left vertical axis. The percentage represents the amount of gated “healthy” cells. Red cells have a varying degree of 7AAD stain. (C) The bar graph shows the percentage of apoptotic cells stained positive with 7AAD in transfected in all experimental groups compared to JEG-3 nontransfected and JEG-3 transfected with a vector without ERAP2. Cytotoxicity assays were run three times with up to three replicates of each experimental groups. The significant cell death observed in JEG-3 cells expressing N392 ERAP2 isoform (*P = 0.040). (D) The table displays the percentage of NK or T cell bound 7AAD positive N392 ERAP2 JEG-3 cells and the significant difference between the two cell types was calculated using the Student t-test (P = 0.0000213). SD, standard deviation.