Increased frequency of human leukocyte antigen–E inhibitory receptor CD94/NKG2A– expressing peritoneal natural killer cells in patients with endometriosis

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Increased frequency of human leukocyte antigen–E inhibitory receptor CD94/NKG2A– expressing peritoneal natural killer cells in patients with endometriosis Ricciarda Galandrini, M.D., Ph.D., Maria Grazia Porpora, M.D., Antonella Stoppacciaro, M.D., Federica Micucci, Ph.D., Cristina Capuano, M.Sc., Ilaria Tassi, Ph.D., Alessia Di Felice, M.D., Pierluigi Benedetti-Panici, M.D., Angela Santoni, Ph.D. Fertility and Sterility Volume 89, Issue 5, Pages 1490-1496 (May 2008) DOI: 10.1016/j.fertnstert.2007.05.018 Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Comparative analysis of CD94/NKG2A and CD94/NKG2C expression in NK cells. The staining patterns of two subjects (control and endometriosis) are shown. Freshly isolated PB and PF mononuclear cells were stained with anti-CD3, anti-CD56, and anti-NKG2A or anti-NKG2C monoclonal antibodies. Samples were analyzed by flow cytometry, and the proportion of NKG2A (A–D) and NKG2C (E–H) were calculated in the gated CD3−, CD56+ population (upper panels). Control subject's PB (B, F) and PF (D, H); endometriotic subject's PB (A, E) and PF (C, G). Fertility and Sterility 2008 89, 1490-1496DOI: (10.1016/j.fertnstert.2007.05.018) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Distribution of CD94/NKG2A on NK cells from the endometriosis and control groups. Freshly isolated PB and PF mononuclear cells were stained with anti-CD3, anti-CD56, and anti-NKG2A or anti-NKG2C monoclonal antibodies. Samples were analyzed by flow cytometry. Graphs of the statistical dot plot showing the percentage of CD94/NKG2A-positive NK cells in the control and endometriosis groups are shown. Each dot represents one subject. Horizontal bars represent the median of the reported values. P values are indicated. Fertility and Sterility 2008 89, 1490-1496DOI: (10.1016/j.fertnstert.2007.05.018) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Endometriotic tissues express HLA-E. Endometriotic specimens of two representative patients were analyzed by reverse-transcription polymerase chain reaction for HLA-E and cytokeratin 20 (CK-20) expression. Total RNA was extracted from whole tissue or from epithelial, inflammatory, and unaffected stromal components upon dissection. Twenty-five–cycle PCR analysis is shown. Fertility and Sterility 2008 89, 1490-1496DOI: (10.1016/j.fertnstert.2007.05.018) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Blockade of CD94/NKG2A receptors restores killing of HLA-E–positive target. Human leukocyte antigen-E–DT360 transfectants and parental HLA-E–negative 721.221 cells were used as targets for PF-derived NK-cell clones in a 4-hour 51Cr release assay. Percentage of specific lysis toward DT360 cells is shown in the presence of anti-NKG2A monoclonal antibodies (black bars) or anti-CD56 control monoclonal antibodies (open bars). Gray bars indicate the percentage of lysis toward 721.221 cells. Three CD94/NKG2A-positive and three CD94/NKG2A-negative NK cell clones derived from a patient with endometriosis are shown. The results are representative of NK cell clones that were derived from two different patients. The effector-target ratio was 2.5:1. Fertility and Sterility 2008 89, 1490-1496DOI: (10.1016/j.fertnstert.2007.05.018) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions