Up-and-down immunity of pregnancy in humans

Unique array of HLA class I molecules on trophoblast

In contrast to most somatic cells, infiltrating EVCT cells do not express the polymorphic T-cell ligands HLA-A and HLA-B class I^16–18 nor do they express HLA class II molecules¹⁷. The absence of polymorphic HLA-A and HLA-B expression thus limits fetal antigen presentation by non-infected EVCT to the maternal CD8⁺ cytotoxic T cells^19,20. Moreover, it has been shown that cytotoxic T cells in the decidua express less perforin and granzyme B than CD8⁺ T cells present in the peripheral blood (PB)^21,22. This is one of the various mechanisms used by the fetus for avoiding killing by CD8⁺ T cells of the invading EVCT which plays a crucial role in uterine vascular remodeling and placental development⁵. EVCT cells express the fetal polymorphic class I molecule, HLA-C, only in the β2-microglobulin-associated form and at a high level^22–24. HLA-C is the ligand of activating or inhibitory killer-cell immunoglobulin receptors (KIRs). Engagement of these inhibitory receptors by specific, and so-far-unknown, trophoblast ligands downregulates the cytotoxic function of dNK cells¹⁵. In addition, EVCT expresses the non-polymorphic HLA-E, HLA-G, and HLA-F^7,25. HLA-E is the ligand of CD94/NKG2A heterodimer inhibitory dNK cell receptor (see next section). HLA-G molecules are expressed at the cell surface of EVCT with a prolonged half-life⁶. HLA-G is the ligand of LILRB1 inhibitory receptor present on about 40% of dNK cells¹⁸. LILRB1 has a high affinity for the dimeric form of HLA-G on EVCT¹⁸. Such strong binding contributes to limit the cytotoxic function of dNK cells²⁶. This is an additional inhibitory mechanism. Syncytiotrophoblast, the outer part of chorionic villi, in contact with the maternal blood of the intervillous space, is devoid of classical and non-classical HLA class I surface expression¹⁷. Thus, it cannot present fetal antigens to the maternal CD8⁺ T cells present in the intervillous space. Though a matter of debate^27,28, some reports indicate that syncytiotrophoblast produces soluble HLA-G^29,30. Soluble HLA-G might exert an immunosuppressive protective role in pregnancy¹⁹ by triggering the killing of maternal activated CD8⁺ T cells present in the decidua basalis as well as in the maternal blood of the intervillous space³¹.

Decidual natural killer cells in healthy pregnancy: lack of cytotoxic function

dNK cells constitute the great majority of maternal immune cells present in early decidua basalis (about 70% of leukocytes present), where the EVCT infiltrates into maternal tissue. The dNK phenotype in healthy pregnancy is unique, differing from PB NK cells¹⁴. The major dNK subpopulation is characterized as CD56^bright/CD16⁻/CD160⁻ (non-cytotoxic phenotype), whereas about 90% of the PB NK cells are CD56^dim/CD16⁺/CD160⁺ (cytotoxic phenotype)³². Although their abundant intracellular lytic granules contain perforin, granzyme, and granulysin³³, dNK cells are poorly cytotoxic⁹. In contrast to circulating NK cells, dNK cells display a poor ability to kill various non-infected cell target lines, including human K562 and .221 as well as murine P815 cell lines³⁴. Other dNK cell functions—regulation of trophoblast invasion and subsequent spiral artery remodeling—are controlled by dNK cell receptors that interact with specific HLA class I or non-major histocompatibility complex (non-MHC) class I ligands present on EVCT³⁵. Similarly, in mice, MHC molecules were shown to educate uterine NK cells to trigger uterine arterial remodeling³⁶. Although dNK cells express several activating receptors, including NKp46, NKp30, NKG2D, and CD94/NKG2C, and some activating receptors of the KIR family, they are not cytotoxic against EVCT¹³. Several studies have characterized the different mechanisms preventing the dNK cells from exerting cytotoxic function. First, this is due to specific interactions between dNK inhibitory receptors and specific ligands. We reported that the engagement of CD94/NKG2A inhibitory receptor with its specific ligand HLA-E expressed by EVCT is a dominant negative regulatory mechanism that prevents cytotoxicity toward trophoblast³⁷. Second, another inhibitory mechanism is provided by engagement of the LILRB1 inhibitory receptor by its specific HLA-G ligand expressed at the cell surface of EVCT³⁸. Third, a report demonstrated that VEGF-C secreted by dNK cells in the first trimester of pregnancy triggered upregulation of TAP-1 in EVCT, protecting them from a cytotoxic role³⁹. Thus, in normal pregnancy, there is an obvious resistance to dNK cell killing function, although dNK cells are potentially capable of cytolytic activity³⁷.

Contact with autologous decidual human cytomegalovirus-infected cells restores decidual natural killer cell killing potential

Human pregnancy is characterized by tolerance to the fetus through down-local decidual immunity but balanced with fetal decidual defense against pathogens. dNK cells are poorly cytotoxic in healthy pregnancy yet possess a likely functional lytic machinery¹⁴. Indeed, using a redirected cell lysis assay, a study reported that specific engagement of NKp46 and, to a lesser extent, NKp30 activating receptors on isolated dNK cells from early decidua induced P815 cell target lysis³⁷. Human cytomegalovirus (HCMV) infection modulates the dNK cell receptor repertoire. Co-culture of dNK cells with HCMV-infected autologous stromal cells increased the number of CD56^dim dNK cells associated with the appearance of CD16 and NKG2C activating receptors⁴⁰. Such a phenotype is consistent with the acquisition of a cytotoxic profile. Similarly, isolated dNK cells infected with Toxoplasma gondii were shown to express markers of cytotoxicity, including increased expression of CD16 and NKG2D receptors, and to acquire a CD56^dim phenotype⁴¹. These different observations indicated that dNK cells acquire cytotoxic potential in the pregnant uterus when the decidua is infected by pathogens. dNK cells become cytotoxic when in contact with HCMV-infected autologous decidual stromal cells⁴². dNK cells engage immune synapse with HCMV-infected autologous stromal cells and polarize their lytic machinery toward infected cells. In contrast, purified dNK cells in contact with uninfected autologous stromal cells do not exert their killing function⁴⁰. Furthermore, via immunohistochemistry, the presence of dNK cells was observed in the vicinity of HCMV-positive cells in placental tissue obtained after elective pregnancy termination⁴⁰. Another article indicated that isolated dNK cells expressing KIR2DS1 activating receptor acquired higher cytotoxic function than KIR2DS1^negative dNK cells when in contact with HCMV-infected decidual stromal cells¹³. Moreover, a recent study reported that a structural modulation of HLA-C is required for a potent KIR2DS1-mediated NK cell activation⁴³. Whether such HLA-C modulation could occur at the cell surface of HCMV-infected EVCT in pregnancy remains to be determined. Thus, dNK cells were shown to clearly exert a cytotoxic function in the pregnant uterus when the decidua is infected by pathogens.

A controversial question can legitimately arise from the above results obtained from in vitro co-culture of isolated dNK cells and stromal cells from the same early pregnancy sample: is there a bias due to the limited type of decidual cells surrounding dNK cells in these in vitro experiments? The beginning of an answer has been provided by a recent article⁴⁴. The authors used a multi-cell-type solid placental tissue containing a variety of immune and non-immune cells, including fetal and maternal cells which more closely resemble infection in vivo. In the latter study, it was demonstrated that HCMV infection triggered a robust interferon-gamma (IFN-γ) and IFN-γ-inducible protein-10 (IP-10) release by dNK cells, thus differing from another study that used in vitro co-culture with only two cell components: autologous dNK and decidual stromal cells⁴⁰. In the latter case, no upregulation of IFN-γ but rather down-modulation of IP-10 by dNK cells was observed⁴⁰. These different results demonstrate the crucial influence of the cellular microenvironment.

Decidual CD8⁺ T cells present in early pregnancy are fully functional¹⁵. They can recognize fetal HLA-C expressed at high levels on the cell surface of EVCT^6,20. HLA-C expressed by EVCT thus exerts a dual role: it can present pathogen-derived peptides to decidual CD8⁺ T cells when EVCT is infected. HLA-C expressed by EVCT is also a specific ligand for various activating KIR dNK receptors²⁴. Such interactions result in the stimulation of dNK cell cytotoxic function¹⁵. Whether virus-specific and HLA-C restricted decidual CD8⁺ T cells are present in infected decidua and able to kill infected cells remains to be determined. Detection of virus-specific CD8⁺ T cells in decidual tissue⁴⁵ suggests that this might be the case. Moreover, a recent study demonstrated that activation of dNK cells through KIR2DS4 activating receptor (which also binds HLA-C) stimulates EVCT migration needed for subsequent placental development⁴⁶.