Natural Killer Cell Education in Women With Recurrent Pregnancy Loss

Abstract

Problem: Natural killer (NK) cells undergo education for full functionality via interactions between killer immunoglobulin-like receptors (KIRs) or NKG2A and human leukocyte antigen (HLA) ligands. Presumably, education is important during early pregnancy as insufficient education has been associated with impaired vascular remodeling and restricted fetal growth in mice. NK cell education is influenced by receptor co-expression patterns, human cytomegalovirus (CMV), the HLA-E^R107^G dimorphism, and HLA-B leader peptide variants. We hypothesized altered NK cell education status and differences in frequencies of HLA-E genotypes and HLA-B leader peptide variants in women with recurrent pregnancy loss (RPL) compared to women with previously uncomplicated pregnancies, and between CMV seropositive and seronegative RPL women.

Methods of study: Peripheral blood mononuclear cells were analyzed by flow cytometry. HLA-ABC was typed by sequence-specific oligonucleotide PCR, and HLA-E by Sanger sequencing. CMV status was determined by anti-CMV IgG immunoassay. NK cells were considered "educated" if the HLA ligand to a KIR or NKG2A was present.

Results: KIR/NKG2A co-expression patterns and percentages of educated NK cells were similar between RPL and controls, and between seropositive and seronegative RPL women. Frequencies of HLA-E genotypes and HLA-B leader peptide variants were comparable. RPL women with the HLA-B T/T variant had a lower percentage of NKG2A-educated NK cells (47.8%) compared to controls (66.4%) (p = 0.025).

Conclusions: HLA-B leader peptide variants might impact NKG2A-specific NK cell education in RPL, warranting validation in larger studies. Follow-up studies are needed to investigate the education status of uterine NK cells and their role in pregnancy.

Keywords: NKG2A; cytomegalovirus; education; killer‐cell immunoglobulin‐like receptor; natural killer cells; recurrent pregnancy loss.

FIGURE 1

Co‐expression pattern of different KIRs in women with RPL and controls. KIR co‐expression pattern on NKG2A⁻ (A) and NKG2A⁺ (B) CD3⁻CD56⁺ NK cells. Bars depict the median with interquartile range; light gray bars indicate controls (n = 16) and dark gray bars indicate women with RPL (n = 45).

FIGURE 2

The impact of anti‐CMV IgG seropositivity on the co‐expression pattern of different KIRs in women with RPL. KIR co‐expression pattern on NKG2A⁻ (A) and NKG2A⁺ (B) CD3⁻CD56⁺ NK cells. Bars display median with interquartile range; gray bars represent the anti‐CMV IgG‐seronegative individuals (n = 12) and red bars the seropositive individuals (n = 15).

FIGURE 3

NK cell education by KIR and NKG2A in women with RPL and controls. Percentages of CD3⁻CD56⁺NKG2A⁻ (A), CD3⁻CD56⁺NKG2A⁺ (B), and total NK cells (CD3⁻CD56⁺NKG2A⁻ plus CD3⁻CD56⁺NKG2A⁺ NK cells) (C). Percentages of CD3⁻CD56⁺ NK cells educated by NKG2A (NKG2A⁺KIR⁻) (D). Single KIR educated: NK cells expressing the combination of KIR2DL1‐C2, KIR2DL2/3‐C1, or KIR3DL1‐Bw4; double KIR educated: NK cells expressing two KIRs in combination with their corresponding ligand; Educated ≥ 1 KIR: NK cells expressing one or more KIRs in combination with their HLA ligand; Uneducated by KIR: NK cells that express no KIRs or KIRs for which the HLA ligand is not genotypically present. NKG2A‐educated: NK cells expressing NKG2A but completely lacking expression of KIR. Dots/boxes depict individual controls (n = 16) and RPL patients (n = 45). Bars indicate median with interquartile range.

FIGURE 4

The impact of anti‐CMV IgG seropositivity on NK cell education by KIR and NKG2A in women with RPL. Percentages of CD3⁻CD56⁺NKG2A⁻ (A), CD3⁻CD56⁺NKG2A⁺ (B), and total NK cells (CD3⁻CD56⁺ NKG2A⁻ plus CD3⁻CD56⁺NKG2A⁺ NK cells) (C). Percentages of CD3⁻CD56⁺ NK cells educated by NKG2A (NKG2A⁺KIR⁻) (D). Single KIR educated: NK cells expressing the combination of KIR2DL1‐C2, KIR2DL2/3‐C1, or KIR3DL1‐Bw4; Educated ≥ 1 KIR: NK cells expressing one or more KIRs in combination with their HLA ligand; uneducated by KIR: NK cells that express no KIRs or KIRs for which the HLA ligand is not genotypically present. NKG2A‐educated: NK cells expressing NKG2A but completely lacking expression of KIR. Every dot represents one individual; gray dots indicate anti‐CMV IgG‐seronegative individuals (n = 12) whereas red dots indicate IgG‐seropositive individuals (n = 15). Bars represent median with interquartile range.

FIGURE 5

The impact of different HLA‐E alleles on the percentage of NKG2A‐educated NK cells in women with RPL versus controls, and in anti‐IgG CMV seropositive versus seronegative women with RPL. Percentages of NKG2A‐educated NK cells according to the different HLA‐E genotypes. Gray dots represent controls (n = 15) and gray boxes depict RPL women (n = 44) (A). Gray dots represent seronegative women with RPL (n = 12) and red dots depict seropositive women with RPL (n = 14) (B). Bars indicate median with interquartile range.

FIGURE 6

The impact of different HLA‐B leader peptide variants on the percentage of NKG2A‐educated NK cells in women with RPL versus controls, and in anti‐CMV IgG seropositive versus seronegative women with RPL. Percentages of NKG2A‐educated NK cells according to the amino acid on position −21 of the leader peptide sequence of HLA‐B; T/T, M/M, or M/T (T = threonine, M = methionine). Gray dots represent controls (n = 16) and gray boxes depict RPL women (n = 45) (A). Gray dots represent seronegative women with RPL (n = 12) and red dots depict seropositive women with RPL (n = 15) (B). Bars indicate median with interquartile range. *p < 0.05.