We previously published a paper reporting the apoptotic nature of first trimester human decidual T cells (Kopcow et al 2008). New experiments and data re-analysis produced confirmatory, negative and contradictory results. We had previously shown by TUNEL and sub diploid DNA content analysis (SD) that ex-vivo decidual T cells, isolated either as CD3+ cells or as CD4+/8+ cells, are apoptotic and that the level of apoptosis differs significantly from peripheral blood T cells isolated in a similar way. Data re-examination including data points previously unintentionally ignored results in indetermination in the dataset structure with contradictory statistical significance results between TUNEL and SD and the two isolation approaches. Furthermore, data disaggregation and granular examination reveals discrepant TUNEL and SD readings within individual samples. We have previously shown that decidual CD3+ lymphocytes stain brighter with AnnexinV -Fitc than peripheral blood T cells and other decidual lympohocyte populations. Independent repetitions with modified and alternative isolation protocols using different flow cytometry equipment in the hands of another researcher failed to reproduce those results, while side by side repetition under the published conditions as well as under the alternative isolation protocol reproduced the published pattern when samples were ran in the same equipment used for the publication. A reflection on these results proposes to understand the scientific paper as one among other possible constructed narratives.

For many years, human peripheral blood natural killer (NK) cells have been divided into functionally distinct CD3- CD56bright CD16- and CD3- CD56dim CD16+ subsets. Recently, several groups of innate lymphoid cells (ILC), distinct from NK cells in development and function, have been defined in mouse. A signature of genes present in mouse ILC except NK cells, defined by Immunological Genome Project studies, is significantly over-represented in human CD56bright cells, by gene set enrichment analysis. Conversely, the signature genes of mouse NK cells are enriched in human CD56dim cells. Correlations are based upon large differences in expression of a few key genes. CD56bright cells show preferential expression of ILC-associated IL7R (CD127), TNFSF10 (TRAIL), KIT (CD117), IL2RA (CD25), CD27, CXCR3, DPP4 (CD26), GPR183, and MHC class II transcripts and proteins. This could indicate an ontological relationship between human CD56bright cells and mouse CD127+ ILC, or conserved networks of transcriptional regulation. In line with the latter hypothesis, among transcription factors known to impact ILC or NK cell development, GATA3, TCF7 (TCF-1), AHR, SOX4, RUNX2, and ZEB1 transcript levels are higher in CD56bright cells, while IKZF3 (AIOLOS), TBX21 (T-bet), NFIL3 (E4BP4), ZEB2, PRDM1 (BLIMP1), and RORA mRNA levels are higher in CD56dim cells.

Decidual NK (dNK) cells, a distinct type of NK cell, are thought to regulate uterine spiral artery remodeling, a process that allows for increased blood delivery to the fetal-placental unit. Impairment of uterine spiral artery remodeling is associated with decreased placental perfusion, increased uterine artery resistance, and obstetric complications such as preeclampsia and intrauterine growth restriction. Ex vivo manipulation of human peripheral blood NK (pNK) cells by a combination of hypoxia, TGFß-1 and 5-aza-2’-deoxycytidine yields cells with phenotypic and in vitro functional similarities to dNK cells, called idNK cells. Here, gene expression profiling shows that CD56Bright idNK cells derived ex vivo from human pNK cells, and to a lesser extent CD56Dim idNK cells, are enriched in the gene expression signature that distinguishes dNK cells from pNK cells. When injected into immunocompromised pregnant mice with elevated uterine artery resistance, idNK cells homed to the uterus and reduced the uterine artery resistance index, suggesting improved placental perfusion.

Natural Killer (NK) cells are key regulators of normal placental development. In contrast to peripheral blood NK (pNK) cells, NK cells located at the maternal-fetal interface constitute the main local lymphocyte population in early pregnancy, are non-cytotoxic and pro-angiogenic. Decidual NK cells (dNK) regulate remodeling of uterine spiral arteries (SA), a process leading to increased blood supply to the developing fetal–placental unit. When SA remodeling is impaired, reduced placental perfusion results in elevated plasma sFlt1 levels, triggering preeclampsia systemic symptoms, (hypertension, edema and proteinuria). We aim to model a NK cell based therapy for preeclampsia. Ex vivo manipulation of human pNK cells by a combination of hypoxia, TGFb-1 and 5-aza-2′-deoxycytidine (AZA) yields cells, termed idNK cells, with phenotypic and in vitro functional similarities to dNK cells including expression of dNK cell surface markers and chemokine receptors, proangiogenic capacity and reduced cytotoxicity. idNK cells were further characterized by microarray gene expression profiling, and their capacity to remodel SA was evaluated in immunodeficient mice presenting narrow SA. Although distinct from dNK cells, idNK cells acquired the gene expression signature that differentiates dNK cells from pNK cells. Most importantly, i-dNK cell injection increased placental perfusion in a mouse model with narrow SA as evidenced by decreased uterine artery resistance evaluated by Doppler ultrasound, and ameliorated SA remodeling. Ex vivo conversion of peripheral blood NK cells into i-dNK cells may be a potential approach for the prevention or treatment of preeclampsia and related disorders. R. de Carvalho Cavalli: None. A. Cerdeira: None. H. Korkes: None. S. Burke: None. A. Rajakumar: None. M. Bhasin: None. S. Karumanchi: Consultant, Commercial Interest: Aggamin LLC. H. Kopcow: None.

Natural Killer (NK) cells are key regulators of normal placental development. In contrast to peripheral blood NK (pNK) cells, NK cells located at the maternal-fetal interface constitute the main local lymphocyte population in early pregnancy, are non-cytotoxic and pro-angiogenic. Decidual NK cells (dNK) regulate remodeling of uterine spiral arteries (SA), a process leading to increased blood supply to the developing fetal-placental unit. When SA remodeling is impaired, reduced placental perfusion results in elevated plasma sFlt1 levels, triggering preeclampsia systemic symptoms, (hypertension, edema and proteinuria). We aim to model a NK cell based therapy for preeclampsia.Ex vivo manipulation of human pNK cells by a combination of hypoxia, TGFb-1 and 5-aza-2'-deoxycytidine (AZA) yields cells, termed idNK cells, with phenotypic and in vitro functional similarities to dNK cells including expression of dNK cell surface markers and chemokine receptors, proangiogenic capacity and reduced cytotoxicity. idNK cells were further characterized by microarray gene expression profiling, and their capacity to remodel SA was evaluated in immunodeficient mice presenting narrow SA.Although distinct from dNK cells, idNK cells acquired the gene expression signature that differentiates dNK cells from pNK cells. Most importantly, i-dNK cell injection increased placental perfusion in a mouse model with narrow SA as evidenced by decreased uterine artery resistance evaluated by Doppler ultrasound, and ameliorated SA remodeling.Ex vivo conversion of peripheral blood NK cells into i-dNK cells may be a potential approach for the prevention or treatment of preeclampsia and related disorders.R. de Carvalho Cavalli: None. A. Cerdeira: None. H. Korkes: None. S. Burke: None. A. Rajakumar: None. M. Bhasin: None. S. Karumanchi: Consultant, Commercial Interest: Aggamin LLC. H. Kopcow: None.

NK cells that populate the decidua are important regulators of normal placentation. In contrast to peripheral blood NK cells, decidual NK (dNK) cells lack cytotoxicity, secrete proangiogenic factors, and regulate trophoblast invasion. In this study we show that exposure to a combination of hypoxia, TGF-β1, and a demethylating agent results in NK cells that express killer cell Ig-like receptors, the dNK cell markers CD9 and CD49a, and a dNK pattern of chemokine receptors. These cells secrete vascular endothelial growth factor (a potent proangiogenic molecule), display reduced cytotoxicity, and promote invasion of human trophoblast cell lines. These findings have potential therapeutic applications for placental disorders associated with altered NK cell biology.

This Commentary highlights the article by Hsu et al (in this issue) reporting an enrichment in induced regulatory T cells (iTregs) in normal pregnancy but not in preeclampsia, implicating iTreg defect as central to the pathogenesis of preeclampsia.

Human NK cells from the decidua basalis of gravid uteri and from the cycling endometrium of women undergoing hysterectomy were isolated and compared by gene expression profiling using Affymetrix microarrays with probes representing ∼47,400 transcripts. Substantial differences indicate that these two types of NK cells represent distinct subsets.

Human peripheral blood NK cells may be divided into two main subsets: CD56 bright CD16 − and CD56 dim CD16 + . Since TGF‐β is known to influence the development of many leukocyte lineages, its effects on NK cell differentiation either from human CD34 + Lin − hematopoietic progenitor/stem cells in vitro or from peripheral blood NK cells were investigated. TGF‐β represses development of NK cells from CD34 + progenitors and inhibits differentiation of CD16 + NK cells. Moreover, TGF‐β also results in conversion of a minor fraction of CD56 bright CD16 + cells found in peripheral blood into CD56 bright CD16 − cells, highlighting a possible role of the former as a developmental intermediate and of TGF‐β in influencing the genesis of NK subsets found in blood.

The human fetus is not rejected by the maternal immune system despite expressing paternal antigens. Natural killer cells, the major lymphocyte population of the human decidua (dNKs), express genes with immunomodulatory potential. These include galectin-1 (gal1), a lectin with apoptotic activity on activated CD8 + T cells, Th1 and Th17 CD4 + cells. Although many cell types at the maternal–fetal interface also produce gal1, its production by dNKs has been used here to study its function in pregnancy. Media conditioned by dNKs containing gal1 induced apoptosis of activated T cells. This effect was blocked by anti-gal1 antibodies. Decidual T (dT) cells but not peripheral T (pT) cells bound gal1 and presented a distinct glycophenotype compatible with sensitivity to gal1. Annexin V staining, TUNEL, and hypodiploidy showed a substantial proportion of apoptotic dT cells. Immunohistochemistry revealed widespread expression of gal1 as well as periglandular apoptotic dT foci that colocalized with dNKs. Thus, secretion of gal1 by dNKs and other decidual cells contributes to the generation of an immune-privileged environment at the maternal–fetal interface.

Mesenchymal stem cells (MSC) based cell transplantation therapy is proved to be an attractive strategy with great potential for improvement of hypoxia induced neural damage. In the present study, MSCs were co-culture with PC12 to investigate its protective effects against hypoxia pretreatment, and the Lactate dehydrogenase (LDH) release assay, MTT and Anexin V staining were performed to analysis the cellular damage or apoptotic. RT-PCR and Western blotting were further used to investigate the underlying mechanism. The results indicate that hypoxia treatment results in the decrease of PC12 cell viability, yet co-culture with MSC could protect the PC12 from hypoxia induced damage. Hypoxia pre-activated or EPO transduced MSC with up-regulated erythropoietin (EPO) expression could further enhance MSC's protective effect against hypoxia induced cell damage, which was associated with high level of anti-apoptotic p-Akt and ration Bcl-2/Bax, and decreased Caspase 3 in PC12. Taken together, these data suggests high levels of MSC-mediated cyto-protection is closely tied to high gene expression levels of EPO. The up-regulation of EPO for enhanced MSC-mediated cyto-protection may has great potential for the MSC cellular therapy of neural or neuronal injuries induced by hypoxia.

Interaction of the activating receptor NKG2D with its ligands is a major stimulatory pathway for cytotoxicity of natural killer (NK) cells. Here, the signaling pathway involved after NKG2D ligation is examined. Either incubation of the NKG2D-bearing human NKL tumor cell line with K562 target cells or cross-linking with NKG2D mAb induced strong activation of the mitogen-activated protein (MAP) kinases. Selective inhibition of JNK MAP kinase with four different means of inhibition greatly reduced NKG2D-mediated cytotoxicity toward target cells and furthermore, blocked the movement of the microtubule organizing center (MTOC), granzyme B (a component of cytotoxic granules), and paxillin (a scaffold protein) to the immune synapse. NKG2D-induced activation of JNK kinase was also blocked by inhibitors of Src protein tyrosine kinases and phospholipase PLCγ, upstream of JNK. Similarly, a second MAP kinase pathway through ERK was previously shown to be required for NK cell cytotoxicity. Thus, activation of two MAP kinase pathways is required for cytotoxic granule and MTOC polarization and for cytotoxicity of human NK cells when NKG2D is ligated.

During pregnancy the uterine decidua is populated by large numbers of natural killer (NK) cells with a phenotype CD56superbrightCD16−CD9+KIR+distinct from both subsets of peripheral blood NK cells. Culture of highly purified CD16+CD9−peripheral blood NK cells in medium containing TGFβ1 resulted in a transition to CD16−CD9+NK cells resembling decidual NK cells. Decidual stromal cells, when isolated and culturedin vitro, were found to produce TGFβ1. Incubation of peripheral blood NK cells with conditioned medium from decidual stromal cells mirrored the effects of TGFβ1. Similar changes may occur upon NK cell entry into the decidua or other tissues expressing substantial TGFβ. In addition, Lin−CD34+CD45+hematopoietic stem/progenitor cells could be isolated from decidual tissue. These progenitors also produced NK cells when cultured in conditioned medium from decidual stromal cells supplemented with IL-15 and stem cell factor.

Activation of natural killer (NK) cell cytotoxicity requires adhesion and formation of a conjugate with a susceptible target cell, followed by actin polymerization, and polarization of the microtubule organizing center (MTOC) and cytolytic granules to the NK cell immune synapse. Here, by using the YTS NK cell line as a model, CD28 is shown to be an activating receptor. It signals cytotoxicity in a process dependent on phosphoinositide-3 kinase activation, leading to sustained extracellular signal-regulated kinase 2 (ERK2) phosphorylation. ERK and phospho-ERK localize to microtubule filaments. Neither conjugation with targets nor actin polymerization is affected by blocking ERK2 activation. However, both polarization of the MTOC and cytolytic granules to the synaptic region and NK cell cytotoxicity are strongly reduced by blocking ERK2 activation. A role for the CD28/CD80 interaction in cytotoxicity of human peripheral NK cells also was established. By contrast, lymphocyte function-associated antigen 1 (LFA-1) ligation transduces only a transient ERK2 activation and fails to induce killing in YTS cells. Thus, in YTS cells, a CD28 signal is used to polarize the MTOC and cytolytic granules to the NK cell immune synapse by stimulating sustained ERK2 activation.

In early pregnancy invading fetal trophoblasts encounter abundant maternal decidual natural killer cells (dNK). dNK express perforin, granzymes A and B and the activating receptors NKp30, NKp44, NKp46, NKG2D, and 2B4 as well as LFA-1. Even though they are granular and express the essential molecules required for lysis, fresh dNK displayed very reduced lytic activity on classical MHC I negative targets K562 and 721.221, ≈15% of that of peripheral NK cells. dNK formed conjugates and activating immune synapses with 721.221 and K562 cells in which CD2, LFA-1 and actin were polarized toward the contact site. However, in contrast to peripheral NK cells, they failed to polarize their microtubule organizing centers and perforin-containing granules to the synapse, accounting for their lack of cytotoxicity.