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In order to dissect the correlation between aberrant TAL1 basic-helix-loop-helix (b-HLH) expression and the exclusive development of T cell acute lymphoblastic leukemias (T-ALL) of the TCRαβ lineage, we have assessed the ability of class A b-HLH proteins to regulate the TCRα and δ enhancers. We demonstrate that E47S binds to TCRα but not to TCRδ E-boxes in vitro. Despite this, neither E2-5 nor HEB transactivate the TCRα enhancer in NIH 3T3, nor did Id1 modify endogenously driven TCRα [αE1-4] activity in a TCRαβ cell line. We also demonstrate that TAL-1 inhibits both binding of E47S to αE3 and αE4 and endogenous transactivation of the TCRα enhancer. Comparison of the activity of the minimal [αE1-2] fragment, which contains no E-boxes, with the accessory [αE3-4] fragment, which contains two, suggested some contribution from the latter to TCRα enhancer activity in HPB-ALL. TCR [αE1-2] activity was partially (40%) inhibited by TAL1 but not all by Id1. In contrast, [αE3-4] activity was almost completely inhibited by TAL1 (80%) and slightly reduced by Id1 (15%). These data demonstrate that class A b-HLH regulation of the TCRα enhancer E-boxes differs from their B lymphoid Igμ counterparts and suggest a novel mechanism on transcriptional inhibition by TAL1, which may be, at least partly, independent of E-box-mediated activation, as we currently recognize it. They also clearly demonstrate that the restriction of TAL1 deregulation to T-ALL of the TCRαβ lineage is not due to induction of TCRα enhancer activity by the TAL1 protein. [ABSTRACT FROM PUBLISHER]

The distinct human leukocyte antigen (HLA) class I expression pattern of human extravillous trophoblasts (EVT) endows them with unique tolerogenic properties that enable successful pregnancy. Nevertheless, how this process is elaborately regulated remains elusive. Previously, E74 like ETS transcription factor 3 (ELF3) was identified to govern high-level HLA-C expression in EVT. In the present study, ELF3 is found to bind to the enhancer region of two adjacent NOD-like receptor (NLR) genes, NLR family pyrin domain-containing 2 and 7 (NLRP2, NLRP7). Notably, our analysis of ELF3-deficient JEG-3 cells, a human choriocarcinoma cell line widely used to study EVT biology, suggests that ELF3 transactivates NLRP7 while suppressing the expression of NLRP2. Moreover, we find that NLRP2 and NLRP7 have opposing effects on HLA-C expression, thus implicating them in immune evasion at the maternal-fetal interface. We confirmed that NLRP2 suppresses HLA-C levels and described a unique role for NLRP7 in promoting HLA-C expression in JEG-3. These results suggest that these two NLR genes, which arose via gene duplication in primates, are fine-tuned by ELF3 yet have acquired divergent functions to enable proper expression levels of HLA-C in EVT, presumably through modulating the degradation kinetics of IkBα. Targeting the ELF3-NLRP2/NLRP7-HLA-C axis may hold therapeutic potential for managing pregnancy-related disorders, such as recurrent hydatidiform moles and fetal growth restriction, and thus improve placental development and pregnancy outcomes., Competing Interests: Competing interests statement:The authors declare no competing interest.

Implantation and maintenance of pregnancy involve intricate immunological processes that enable the developing fetus to coexist with the maternal immune system. Progesterone, a critical hormone during pregnancy, is known to promote immune tolerance and prevent preterm labor. However, the mechanism by which progesterone mediates these effects remains unclear. In this study, we investigated the role of the non-classical progesterone receptor membrane component 1 (PGRMC1) in progesterone signaling at the maternal-fetal interface. Using JEG3 cells, a trophoblast model cell line, we observed that progesterone stimulation increased the expression of human leukocyte antigen-C (HLA-C) and HLA-G, key molecules involved in immune tolerance. We also found that progesterone upregulated the expression of the transcription factor ELF3, which is known to regulate trophoblast-specific HLA-C expression. Interestingly, JEG3 cells lacked expression of classical progesterone receptors (PRs) but exhibited high expression of PGRMC1, a finding we confirmed in primary trophoblasts by mining sc-RNA seq data from human placenta. To investigate the role of PGRMC1 in progesterone signaling, we used CRISPR/Cas9 technology to knockout PGRMC1 in JEG3 cells. PGRMC1-deficient cells showed a diminished response to progesterone stimulation. Furthermore, we found that the progesterone antagonist RU486 inhibited ELF3 expression in a PGRMC1-dependent manner, suggesting that RU486 acts as a progesterone antagonist by competing for receptor binding. Additionally, we found that RU486 inhibited cell invasion, an important process for successful pregnancy, and this inhibitory effect was dependent on PGRMC1. Our findings highlight the crucial role of PGRMC1 in mediating the immunoregulatory effects of progesterone at the maternal-fetal interface., Competing Interests: Declaration of Competing Interest The authors report no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Zika virus (ZIKV) during pregnancy infects fetal trophoblasts and causes placental damage and birth defects including microcephaly. Little is known about the anti-ZIKV cellular immune response at the maternal-fetal interface. Decidual natural killer cells (dNK), which directly contact fetal trophoblasts, are the dominant maternal immune cells in the first-trimester placenta, when ZIKV infection is most hazardous. Although dNK express all the cytolytic molecules needed to kill, they usually do not kill infected fetal cells but promote placentation. Here, we show that dNK degranulate and kill ZIKV-infected placental trophoblasts. ZIKV infection of trophoblasts causes endoplasmic reticulum (ER) stress, which makes them dNK targets by down-regulating HLA-C/G, natural killer (NK) inhibitory receptor ligands that help maintain tolerance of the semiallogeneic fetus. ER stress also activates the NK activating receptor NKp46. ZIKV infection of Ifnar1 -/- pregnant mice results in high viral titers and severe intrauterine growth restriction, which are exacerbated by depletion of NK or CD8 T cells, indicating that killer lymphocytes, on balance, protect the fetus from ZIKV by eliminating infected cells and reducing the spread of infection., Competing Interests: The authors declare no competing interest.

Humanized double transgenic mice express both HLA-DR15 (the MHC gene linked to MS) and TCR.Ob1A12 from a multiple sclerosis patient (that recognizes MBP85-99 presented by HLA-DR15), yet they fail to develop autoimmune encephalomyelitis quickly, although 5-10% develop disease at 12 months. These mice were found to express large numbers of IL-10-secreting splenocytes as early as 4 weeks of age. These regulatory T cells appeared spontaneously without prior immunization with the autoantigen MBP85-99. They were of murine origin and had a cytokine secretion profile and surface phenotype similar to that reported for Tr1 cells. Notably, the frequency of disease appeared to increase at 14 months. The diseased mice had small spleens which averaged 47 mg, while the remaining non-diseased mice in our colony killed at ages 14-15 months had splenocytes that averaged 80 mg (ranging from 47-130 mg). Thus, the appearance of disease was associated with diminution in numbers of IL-10-secreting regulatory T cells with age., (© 2021 John Wiley & Sons Ltd.)

HLA-C arose during evolution of pregnancy in the great apes 10 to 15 million years ago. It has a dual function on placental extravillous trophoblasts (EVTs) as it contributes to both tolerance and immunity at the maternal-fetal interface. The mode of its regulation is of considerable interest in connection with the biology of pregnancy and pregnancy abnormalities. First-trimester primary EVTs in which HLA-C is highly expressed, as well as JEG3, an EVT model cell line, were employed. Single-cell RNA-seq data and quantitative PCR identified high expression of the transcription factor ELF3 in those cells. Chromatin immunoprecipitation (ChIP)-PCR confirmed that both ELF3 and MED1 bound to the proximal HLA-C promoter region. However, binding of RFX5 to this region was absent or severely reduced, and the adjacent HLA-B locus remained closed. Expression of HLA-C was inhibited by ELF3 small interfering RNAs (siRNAs) and by wrenchnolol treatment. Wrenchnolol is a cell-permeable synthetic organic molecule that mimics ELF3 and is relatively specific for binding to ELF3's coactivator, MED23, as our data also showed in JEG3. Moreover, the ELF3 gene is regulated by a superenhancer that spans more than 5 Mb, identified by assay for transposase-accessible chromatin using sequencing (ATAC-seq), as well as by its sensitivity to (+)-JQ1 (inhibitor of BRD4). ELF3 bound to its own promoter, thus creating an autoregulatory feedback loop that establishes expression of ELF3 and HLA-C in trophoblasts. Wrenchnolol blocked binding of MED23 to ELF3, thus disrupting the positive-feedback loop that drives ELF3 expression, with down-regulation of HLA-C expression as a consequence., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Maternal decidual NK (dNK) cells promote placentation, but how they protect against placental infection while maintaining fetal tolerance is unclear. Here we show that human dNK cells highly express the antimicrobial peptide granulysin (GNLY) and selectively transfer it via nanotubes to extravillous trophoblasts to kill intracellular Listeria monocytogenes (Lm) without killing the trophoblast. Transfer of GNLY, but not other cell death-inducing cytotoxic granule proteins, strongly inhibits Lm in human placental cultures and in mouse and human trophoblast cell lines. Placental and fetal Lm loads are lower and pregnancy success is greatly improved in pregnant Lm-infected GNLY-transgenic mice than in wild-type mice that lack GNLY. This immune defense is not restricted to pregnancy; peripheral NK (pNK) cells also transfer GNLY to kill bacteria in macrophages and dendritic cells without killing the host cell. Nanotube transfer of GNLY allows dNK to protect against infection while leaving the maternal-fetal barrier intact., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

During pregnancy, invading HLA-G+ extravillous trophoblasts (EVT) play a key role in placental development, uterine spiral artery remodeling, and prevention of detrimental maternal immune responses to placental and fetal antigens. Failures of these processes are suggested to play a role in the development of pregnancy complications, but very little is known about the underlying mechanisms. Here we present validated methods to purify and culture primary HLA-G+ EVT from the placental disk and chorionic membrane from healthy term pregnancy. Characterization of HLA-G+ EVT from term pregnancy compared to first trimester revealed their unique phenotypes, gene expression profiles, and differing capacities to increase regulatory T cells (Treg) during coculture assays, features that cannot be captured by using surrogate cell lines or animal models. Furthermore, clinical variables including gestational age and fetal sex significantly influenced EVT biology and function. These methods and approaches form a solid basis for further investigation of the role of HLA-G+ EVT in the development of detrimental placental inflammatory responses associated with pregnancy complications, including spontaneous preterm delivery and preeclampsia., Competing Interests: The authors declare no competing interest.

Decidual NK cells (dNK) are the main lymphocyte population in early pregnancy decidual mucosa. Although dNK decrease during pregnancy, they remain present in decidual tissues at term. First trimester dNK facilitate trophoblast invasion, provide protection against infections, and were shown to have many differences in their expression of NKRs, cytokines, and cytolytic capacity compared with peripheral blood NK cells (pNK). However, only limited data are available on the phenotype and function of term pregnancy dNK. In this study, dNK from human term pregnancy decidua basalis and decidua parietalis tissues were compared with pNK and first trimester dNK. Profound differences were found, including: 1) term pregnancy dNK have an increased degranulation response to K562 and PMA/ionomycin but lower capacity to respond to human CMV-infected cells; 2) term pregnancy dNK are not skewed toward recognition of HLA-C, as was previously shown for first trimester dNK; and 3) protein and gene expression profiles identified multiple differences between pNK, first trimester, and term pregnancy dNK, suggesting term pregnancy dNK are a distinct type of NK cells. Understanding the role of dNK throughout pregnancy is of high clinical relevance for studies aiming to prevent placental inflammatory disorders as well as maternal-to-fetal transmission of pathogens., (Copyright © 2020 by The American Association of Immunologists, Inc.)

To establish a healthy pregnancy, maternal immune cells must tolerate fetal allo-antigens and remain competent to respond to infections both systemically and in placental tissues. Extravillous trophoblasts (EVT) are the most invasive cells of extra-embryonic origin to invade uterine tissues and express polymorphic Human Leucocyte Antigen-C (HLA-C) of both maternal and paternal origin. Thus, HLA-C is a key molecule that can elicit allogeneic immune responses by maternal T and NK cells and for which maternal-fetal immune tolerance needs to be established. HLA-C is also the only classical MHC molecule expressed by EVT that can present a wide variety of peptides to maternal memory T cells and establish protective immunity. The expression of paternal HLA-C by EVT provides a target for maternal NK and T cells, whereas HLA-C expression levels may influence how this response is shaped. This dual function of HLA-C requires tight transcriptional regulation of its expression to balance induction of tolerance and immunity. Here, we critically review new insights into: (i) the mechanisms controlling expression of HLA-C by EVT, (ii) the mechanisms by which decidual NK cells, effector T cells and regulatory T cells recognize HLA-C allo-antigens, and (iii) immune recognition of pathogen derived antigens in context of HLA-C., (Copyright © 2019 Papúchová, Meissner, Li, Strominger and Tilburgs.)

During pregnancy, maternal regulatory T cells (Tregs) are important in establishing immune tolerance to invading fetal extravillous trophoblasts (EVTs). CD25 HI FOXP3+ Tregs are found at high levels in decidual tissues and have been shown to suppress fetus-specific and nonspecific responses. However, limited data are available on additional decidual Treg types and the mechanisms by which they are induced. This study investigated three distinct decidual CD4+ Treg types in healthy pregnancies with a regulatory phenotype and the ability to suppress T cell responses: CD25 HI FOXP3+, PD1 HI IL-10+, and TIGIT+FOXP3 dim . Moreover, co-culture of HLA-G+ EVTs or decidual macrophages with blood CD4+ T cells directly increased the proportions of CD25 HI FOXP3+ Tregs compared to T cells cultured alone. EVTs also increased PD1 HI Tregs that could be inhibited by HLA-C and CD3 antibodies, suggesting an antigen-specific induction. The presence of distinct Treg types may allow for the modulation of a variety of inflammatory responses in the placenta., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Polymorphic HLAs form the primary immune barrier to cell therapy. In addition, innate immune surveillance impacts cell engraftment, yet a strategy to control both, adaptive and innate immunity, is lacking. Here we employed multiplex genome editing to specifically ablate the expression of the highly polymorphic HLA-A/-B/-C and HLA class II in human pluripotent stem cells. Furthermore, to prevent innate immune rejection and further suppress adaptive immune responses, we expressed the immunomodulatory factors PD-L1, HLA-G, and the macrophage "don't-eat me" signal CD47 from the AAVS1 safe harbor locus. Utilizing in vitro and in vivo immunoassays, we found that T cell responses were blunted. Moreover, NK cell killing and macrophage engulfment of our engineered cells were minimal. Our results describe an approach that effectively targets adaptive as well as innate immune responses and may therefore enable cell therapy on a broader scale., Competing Interests: Conflict of interest statement: C.A.C. is a founder and chief scientific officer of Sana Biotechnology. D.A.M. is a scientific founder and a board observer of Semma Therapeutics. Neither a reagent nor any funding from these companies was used in this study. Harvard University has filed patent applications that cover these inventions.

Random amino acid copolymers used in the treatment of multiple sclerosis in man or experimental autoimmune encephalomyelitis (EAE) in mice [poly(Y,E,A,K) n , known as Copaxone, and poly(Y,F,A,K) n ] function at least in part by generation of IL-10-secreting regulatory T cells that mediate bystander immunosuppression. The mechanism through which these copolymers induce Tregs is unknown. To investigate this question, four previously described Vα3.2 Vβ14 T cell receptor (TCR) cDNAs, the dominant clonotype generated in splenocytes after immunization of SJL mice, that differed only in their CDR3 sequences were utilized to generate retrogenic mice. The high-level production of IL-10 as well as IL-5 and small amounts of the related cytokines IL-4 and IL-13 by CD4 + T cells isolated from the splenocytes of these mice strongly suggests that the TCR itself encodes information for specific cytokine secretion. The proliferation and production of IL-10 by these Tregs was costimulated by activation of glucocorticoid-induced TNF receptor (GITR) (expressed at high levels by these cells) through its ligand GITRL. A mechanism for generation of cells with this specificity is proposed. Moreover, retrogenic mice expressing these Tregs were protected from induction of EAE by the appropriate autoantigen., Competing Interests: The authors declare no conflict of interest.

Understanding how decidual CD8 + T cell (CD8 + dT) cytotoxicity is regulated and how these cells integrate the competing needs for maternal-fetal tolerance and immunity to infection is an important research and clinical goal. Gene-expression analysis of effector-memory CD8 + dT demonstrated a mixed transcriptional signature of T cell dysfunction, activation, and effector function. High protein expression of coinhibitory molecules PD1, CTLA4, and LAG3, accompanied by low expression of cytolytic molecules suggests that the decidual microenvironment reduces CD8 + dT effector responses to maintain tolerance to fetal antigens. However, CD8 + dT degranulated, proliferated, and produced IFN-γ, TNF-α, perforin, and granzymes upon in vitro stimulation, demonstrating that CD8 + dT are not permanently suppressed and retain the capacity to respond to proinflammatory events, such as infections. The balance between transient dysfunction of CD8 + dT that are permissive of placental and fetal development, and reversal of this dysfunctional state, is crucial in understanding the etiology of pregnancy complications and prevention of congenital infections., Competing Interests: The authors declare no conflict of interest.

For many years, human peripheral blood natural killer (NK) cells have been divided into functionally distinct CD3 - CD56 bright CD16 - and CD3 - CD56 dim 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 CD56 bright cells, by gene set enrichment analysis. Conversely, the signature genes of mouse NK cells are enriched in human CD56 dim cells. Correlations are based upon large differences in expression of a few key genes. CD56 bright 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 CD56 bright 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 CD56 bright cells, while IKZF3 (AIOLOS), TBX21 (T-bet), NFIL3 (E4BP4), ZEB2, PRDM1 (BLIMP1), and RORA mRNA levels are higher in CD56 dim cells.

During pregnancy, fetal extravillous trophoblasts (EVT) play a key role in the regulation of maternal T cell and NK cell responses. EVT display a unique combination of human leukocyte antigens (HLA); EVT do not express HLA-A and HLA-B, but do express HLA-C, HLA-E, and HLA-G. The mechanisms establishing this unique HLA expression pattern have not been fully elucidated. The major histocompatibility complex (MHC) class I and class II transcriptional activators NLRC5 and CIITA are expressed neither by EVT nor by the EVT model cell line JEG3, which has an MHC expression pattern identical to that of EVT. Therefore, other MHC regulators must be present to control HLA-C, HLA-E, and HLA-G expression in these cells. CIITA and NLRC5 are both members of the nucleotide-binding domain, leucine-rich repeat (NLR) family of proteins. Another member of this family, NLRP2, is highly expressed by EVT and JEG3, but not in maternal decidual stromal cells. In this study, transcription activator-like effector nuclease technology was used to delete NLRP2 in JEG3. Furthermore, lentiviral delivery of shRNA was used to knockdown NLRP2 in JEG3 and primary EVT. Upon NLRP2 deletion, Tumor Necrosis Factor-α (TNFα)-induced phosphorylation of NF-KB p65 increased in JEG3 and EVT, and more surprisingly a significant increase in constitutive HLA-C expression was observed in JEG3. These data suggest a broader role for NLR family members in the regulation of MHC expression during inflammation, thus forming a bridge between innate and adaptive immune responses. As suppressor of proinflammatory responses, NLRP2 may contribute to preventing unwanted antifetal responses., (© The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please journals.permissions@oup.com.)

During pregnancy, semiallogeneic fetal extravillous trophoblasts (EVT) invade the uterine mucosa without being rejected by the maternal immune system. Several mechanisms were initially proposed by Peter Medawar half a century ago to explain this apparent violation of the laws of transplantation. Then, three decades ago, an unusual human leukocyte antigen (HLA) molecule was identified: HLA-G. Uniquely expressed in EVT, HLA-G has since become the center of the present understanding of fetus-induced immune tolerance. Despite slow progress in the field, the last few years have seen an explosion in our knowledge of HLA-G biology. Here, we critically review new insights into the mechanisms controlling the expression and function of HLA-G at the maternal-fetal interface, and discuss their relevance for fetal tolerance., (Copyright © 2017. Published by Elsevier Ltd.)

To establish a healthy pregnancy the maternal immune system must tolerate fetal allo-antigens, yet remain competent to respond to infections. The ability of decidual NK cells (dNK) to promote migration of fetal extravillous trophoblasts (EVT) and placental growth as well as the capacity of EVT to promote immune tolerance are topics of high interest and extensive research. However, the problem of how dNK and decidual CD8+ T cells (CD8+ dT) provide immunity to infections of the placenta and the mechanisms that regulate their cytolytic function has thus far largely been ignored. Fetal EVT are the most invasive cells of the placenta and directly interact with maternal decidual immune cells at this maternal-fetal interface. Besides the expression of non-polymorphic HLA-E and HLA-G molecules that are associated with immune tolerance, EVT also express highly polymorphic HLA-C molecules that can serve as targets for maternal dNK and CD8+ dT responses. HLA-C expression by EVT has a dual role as the main molecule to which immune tolerance needs to be established and as the only molecule that can present pathogen-derived peptides and provide protective immunity when EVT are infected. The focus of this review is to address the regulation of cytotoxicity of dNK and CD8+ dT, which is essential for maternal-fetal immune tolerance as well as recent evidence that both cell types can provide immunity to infections at the maternal-fetal interface. A particular emphasis is given to the role of HLA-C expressed by EVT and its capacity to elicit dNK and CD8+ dT responses., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The combination of the activating killer cell Ig-like receptor 2DS1 (KIR2DS1) expressed by maternal decidual natural killer cells (dNK) and the presence of its ligand, the HLA-C allotype HLA-C2, expressed by fetal trophoblasts, reduces the risk of developing pregnancy complications. However, no molecular or cellular mechanism explains this genetic correlation. Here we demonstrate that KIR2DS1+ dNK acquired higher cytotoxic function than KIR2DS1- dNK when exposed to human cytomegalovirus (HCMV)-infected decidual stromal cells (DSC), particularly when DSCs express HLA-C2. Furthermore, dNK were unable to degranulate or secrete cytokines in response to HCMV-infected primary fetal extravillous trophoblasts. This emphasizes the immunological challenge to clear placental viral infections within the immune-privileged placenta. Activation of dNK through KIR2DS1/HLA-C2 interaction increases their ability to respond to placental HCMV infection and may limit subsequent virus-induced placental pathology. This mechanism is directly related to how KIR2DS1 expressed by dNK reduces development of severe pregnancy complications such as miscarriages and preterm delivery., Competing Interests: The authors declare no conflict of interest.

HLA-G, a nonclassical HLA molecule uniquely expressed in the placenta, is a central component of fetus-induced immune tolerance during pregnancy. The tissue-specific expression of HLA-G, however, remains poorly understood. Here, systematic interrogation of the HLA-G locus using massively parallel reporter assay (MPRA) uncovered a previously unidentified cis-regulatory element 12 kb upstream of HLA-G with enhancer activity, Enhancer L Strikingly, clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of this enhancer resulted in ablation of HLA-G expression in JEG3 cells and in primary human trophoblasts isolated from placenta. RNA-seq analysis demonstrated that Enhancer L specifically controls HLA-G expression. Moreover, DNase-seq and chromatin conformation capture (3C) defined Enhancer L as a cell type-specific enhancer that loops into the HLA-G promoter. Interestingly, MPRA-based saturation mutagenesis of Enhancer L identified motifs for transcription factors of the CEBP and GATA families essential for placentation. These factors associate with Enhancer L and regulate HLA-G expression. Our findings identify long-range chromatin looping mediated by core trophoblast transcription factors as the mechanism controlling tissue-specific HLA-G expression at the maternal-fetal interface. More broadly, these results establish the combination of MPRA and CRISPR/Cas9 deletion as a powerful strategy to investigate human immune gene regulation.

The interaction of noncytotoxic decidual natural killer cells (dNK) and extravillous trophoblasts (EVT) at the maternal-fetal interface was studied. Confocal microscopy revealed that many dNK interact with a single large EVT. Filamentous projections from EVT enriched in HLA-G were shown to contact dNK, and may represent the initial stage of synapse formation. As isolated, 2.5% of dNK contained surface HLA-G. However, surface HLA-G-negative dNK contained internalized HLA-G. Activation of dNK resulted in the disappearance of internalized HLA-G in parallel with restoration of cytotoxicity. Surface HLA-G was reacquired by incubation with EVT. This HLA-G cycle of trogocytosis, endocytosis, degradation, and finally reacquisition provides a transient and localized acquisition of new functional properties by dNK upon interaction with EVT. Interruption of the cycle by activation of dNK by cytokines and/or viral products serves to ensure the NK control of virus infection at the interface, and is illustrated here by the response of dNK to human cytomegalo virus (HCMV)-infected decidual stromal cells. Thus, the HLA-G cycle in dNK can provide both for NK tolerance and antiviral immunity.

Problem: CD11c(HI) human decidual macrophages express several isoforms of CD1 molecules. Their expression pattern and function required investigation., Method of Study: CD11c(HI) macrophages were isolated from decidua. Expression of CD1 isoforms and their ability to present lipid antigens to T cells was studied., Results: CD1a, CD1c, and CD1d were all expressed on CD11c(HI) dMϕ, a pattern differing from those previously observed. Exposure of peripheral monocytes and dendritic cells to lipid isolates from decidua led to increased surface CD1a levels only. The CD1a and CD1c on dMϕ were able to present the appropriate lipid antigens to lipid antigen-specific T cells. Finally, autoreactivity of decidual T cells to CD1a was observed., Conclusion: The unique pattern of expression of CD1 isoforms on CD11c(HI) dMϕ is consistent with organ-specific roles of CD1 in human T-cell responses. dMϕ are able to present lipid antigens to both peripheral and decidual T cells and are major antigen-presenting cells in human decidua., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Invading human leukocyte antigen-G+ (HLA-G+) extravillous trophoblasts (EVT) are rare cells that are believed to play a key role in the prevention of a maternal immune attack on foreign fetal tissues. Here highly purified HLA-G+ EVT and HLA-G- villous trophoblasts (VT) were isolated. Culture on fibronectin that EVT encounter on invading the uterus increased HLA-G, EGF-Receptor-2, and LIF-Receptor expression on EVT, presumably representing a further differentiation state. Microarray and functional gene set enrichment analysis revealed a striking immune-activating potential for EVT that was absent in VT. Cocultures of HLA-G+ EVT with sample matched decidual natural killer cells (dNK), macrophages, and CD4+ and CD8+ T cells were established. Interaction of EVT with CD4+ T cells resulted in increased numbers of CD4+CD25(HI)FOXP3+CD45RA+ resting regulatory T cells (Treg) and increased the expression level of the Treg-specific transcription factor FOXP3 in these cells. However, EVT did not enhance cytokine secretion in dNK, whereas stimulation of dNK with mitogens or classical natural killer targets confirmed the distinct cytokine secretion profiles of dNK and peripheral blood NK cells (pNK). EVT are specialized cells involved in maternal-fetal tolerance, the properties of which are not imitated by HLA-G-expressing surrogate cell lines.

The reciprocal activation of dendritic cells (DCs) and natural killer cells (NKs) plays a key role in both innate and adaptive immunity. The effect of aging on this cross-talk, a critical step in virus disease control and tumor immunology, has not been reported. Splenic DCs and NKs were purified from both young and old C57BL/6 mice and cocultured in the presence of polyinosinic:polycytidylic acid (poly I:C). The resulting activation of NKs was measured as expression of CD69 and secretion of IFN-γ. However, DCs from old mice could not activate NKs from either young or old mice in vitro or in vivo. In contrast, DCs from young mice efficiently activated NKs from both young and old mice. DCs from old mice were deficient in poly I:C-stimulated secretion of IL-15, IL-18, and IFN-α. Gene expression analysis revealed many other differences between DCs of old and young mice. Young mice strongly eradicated MHC class I-negative NK-sensitive RMA-S lymphoma mutant tumor cells, but old mice did not, in concert with the previous report that mousepox kills aged, but not young, C57BL/6 mice. Furthermore, a similar dysfunction of DC and its key role in NK activation was found in 27 out of 55 healthy human donors.

Regulatory T cells (Tregs) play crucial roles in both fetal and tumor development. We recently showed that immunosurveillance by pre-existing CD44(high)CD62L(low) activated/memory Tregs (amTregs) specific for self-Ags protects emergent tumor cells in mice. This Treg response of a memory type is more rapid than and dominates the antitumor response of tumor-specific effector T cells. In this study, we report striking similarities between the early Treg responses to embryo and tumor implantation. Tregs are rapidly recruited to uterus-draining lymph nodes and activated in the first days after embryo implantation in both syngeneic and allogeneic matings; express the markers of the amTreg subset; and are at least in part self-Ag specific, as seen in tumor emergence. Unlike in the tumor emergence setting, however, for which preimmunization against tumor Ags is sufficient for complete tumor eradication even in the presence of Tregs, Treg depletion is additionally required for high frequencies of fetus loss after preimmunization against paternal tissue Ags. Thus, amTregs play a major role in protecting embryos in both naive and preimmune settings. This role and the ensuing therapeutic potential are further highlighted by showing that Treg stimulation, directly by low-dose IL-2 or indirectly by Fms-related tyrosine kinase 3 ligand, led to normal pregnancy rates in a spontaneous abortion-prone model.

Multiple sclerosis (MS) is an autoimmune disease that affects the CNS. One approved treatment for relapsing forms of MS is YEAK, a random copolymer of the amino acids tyrosine, glutamic acid, alanine, and lysine. YFAK, a second-generation copolymer composed of tyrosine, phenylalanine, alanine, and lysine, is more successful in treating experimental autoimmune encephalomyelitis, a mouse model of MS. Although originally designed and optimized based on the autoantigen myelin basic protein (MBP) and the MBP-derived peptide MBP85-99 presented to the MS-associated class II MHC molecule HLA-DR2, YEAK and YFAK also stimulate cytokine and chemokine production in APCs that lack class II MHC products. How YEAK and YFAK copolymers interact with APCs remains enigmatic. We used biotinylated YFAK to affinity-purify YFAK-interacting proteins from RAW264.7 cells and tested APCs from mice deficient in several of the newly identified interactors for their capacity to secrete CCL22 in response to YEAK and YFAK. We propose that initial contact of YFAK with cells is mediated mainly by electrostatic interactions, and find that interaction of YFAK with host proteins is strongly dependent on ionic strength. Cells deficient in enzymes involved in sulfation of proteins and proteoglycans showed strongly reduced binding of biotinylated YFAK. Lastly, cells stimulated with YFAK in the presence of heparin, structurally similar to heparan sulfates, failed to produce CCL22. We conclude that charge-dependent interactions of copolymers that alleviate MS/experimental autoimmune encephalomyelitis are critical for their effects exerted on APCs and may well be the main initial mediators of these therapeutically active copolymers.

During pregnancy CD8+ effector T cells need optimal immune regulation to prevent a detrimental response to allogeneic fetal cells while providing immune protection to infections. A significant proportion of (prospective) mothers carry naïve or memory CD8+ T cells with a TCR that can directly bind to paternal MHC molecules. In addition, a high percentage of pregnant women develop specific T cell responses to fetal minor histocompatibility antigens (mHags). Under normal conditions, fetal-maternal MHC and mHag mismatches lead to elevated lymphocyte activation but do not induce pregnancy failure. Furthermore, viral infections alter the maternal CD8+ T cell response by changing the CD8+ T cell repertoire and increasing the influx of CD8+ T cells to decidual tissue. The normally high T cell activation threshold at the fetal-maternal interface may prevent efficient clearance of viral infections. Conversely, the increased inflammatory response due to viral infections may break fetal-maternal tolerance and lead to pregnancy complications. The aim of this review is to discuss the recent studies of CD8+ T cells in pregnancy, identify potential mechanisms for antigen-specific immune recognition of fetal extravillous trophoblast (EVT) cells by CD8+ T cells, and discuss the impact of viral infections and virus-specific CD8+ T cells during pregnancy., (© 2013 John Wiley & Sons A/S.)

The effects of the amino acid copolymers used in the therapy of experimental autoimmune encephalomyelitis, poly(Y,E,A,K)(n) (Copaxone(®)) and poly(Y,F,A,K)(n), on murine myeloid cells have been investigated. After administration of these copolymers to mice, increases in several splenic myeloid cell populations were observed, including CD11b(+) CD11c(+) dendritic cells. The latter were the major splenic cell type that secreted CCL22 (macrophage-derived chemokine) on stimulation with amino acid copolymers. CCL22 secretion was also stimulated from bone marrow-derived dendritic cells (BMDC) generated with GM-CSF in much larger amounts than from bone marrow-derived macrophages generated with M-CSF. Moreover, CCL22 secretion could also be obtained using BMDC generated from two different types of MHC II(-/-) mice, indicating that an innate immune receptor is involved. Finally, incubation of these BMDC or splenic dendritic cells with naive CD4(+) CD25(-) T cells resulted in formation of CD4(+) CD25(HI) Foxp3 T cells (~25% of which were Foxp3(+)). The number of these regulatory cells was doubled by pretreatment of BMDC with amino acid copolymers.

NK cells eliminate cancer and virus-infected cells through their cytolytic activity. The last step in NK-cell cytotoxicity, resulting in exocytosis of granule content, requires fusion of lytic granules with the plasma membrane. Proteins from the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family mediate membrane fusion events in the cell. Here, we show that NK cells express all members of the R-SNARE subgroup. Two of these R-SNARE proteins, VAMP4 and VAMP7, colocalize with lytic granules during cytotoxic interactions. However, only VAMP7 associates with perforin-containing granules in nonactivated cells, indicating that the two VAMPs have different functions in exocytosis. Using both the tumor NK-cell line YTS and the peripheral NK cells, we show that the disruption of expression of either VAMP4 or VAMP7 inhibits the release of lytic granules and severely impairs NK-cell cytotoxic activity. Furthermore, VAMP7 but not VAMP4 is involved in IFN-γ secretion in NK cells, indicating that VAMP7 is involved in many fusion processes and thus plays a more general function in NK-cell activity than VAMP4., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Several important events occur at the maternal-fetal interface, including generation of maternal-fetal tolerance, remodeling of the uterine smooth muscle and its spiral arteries and glands, and placental construction. Fetal-derived extravillous trophoblasts come in direct contact with maternal decidual leukocytes. Macrophages represent ∼20% of the leukocytes at this interface. In this study, two distinct subsets of CD14(+) decidual macrophages (dMs) are found to be present in first-trimester decidual tissue, CD11c(HI) and CD11c(LO). Gene expression analysis by RNA microarray revealed that 379 probes were differentially expressed between these two populations. Analysis of the two subsets revealed several clusters of coregulated genes that suggest distinct functions for these subsets in tissue remodeling, growth, and development. CD11c(HI) dMs express genes associated with lipid metabolism and inflammation, whereas CD11c(LO) dMs express genes associated with extracellular matrix formation, muscle regulation, and tissue growth. The CD11c(HI) dMs also differ from CD11c(LO) dMs in their ability to process protein Ag and are likely to be the major APCs in the decidua. Moreover, these populations each secrete both proinflammatory and anti-inflammatory cytokines that may contribute to the balance that establishes fetal-maternal tolerance. Thus, they do not fit the conventional M1/M2 categorization.

The random amino acid copolymer poly(Y,E,A,K)(n) (Copaxone®) is widely used in multiple sclerosis treatment and a second generation copolymer poly(Y,F,A,K)(n) with enhanced efficacy in experimental autoimmune encephalomyelitis in mice has been described. A major mechanism through which copolymers function to ameliorate disease is the generation of immunosuppressive IL-10-secreting regulatory T cells entering the CNS. In addition, the antigen presenting cell to which these copolymers bind through MHC Class II proteins may have an important role. Here, both CCL22 (a Th2 cell chemoattractant) in large amounts and CXCL13 in much smaller amounts are shown to be secreted after administration of YFAK to mice and to a smaller extent by YEAK parallel to their serum concentrations. Moreover, bone marrow-derived macrophages secrete CCL22 in vitro in response to YFAK and to higher concentrations of YEAK. Strikingly, these chemokines are also secreted into serum of MHC Class II -/- mice, indicating that an innate immune receptor on these cells also has an important role. Thus, both the innate and the adaptive immune systems are involved in the mechanism of EAE amelioration by YFAK. The enhanced ability of YFAK to stimulate the innate immune system may account for its enhanced efficacy in EAE treatment.

The generation of T cell receptor (TCR) sequence diversity can produce 'forbidden' clones able to recognize self-antigens. Here, the structure of the complex between a myelin basic protein peptide (MBP85-99), human leukocyte antigen (HLA)-DR2 (DRB1*1501/DRA) and TCR-Ob.2F3, the dominant autoimmune clone obtained from a multiple sclerosis (MS) patient, has been determined using structural docking simulation and dynamics in silico and compared to the structure of TCR-Ob.1A12 complexes with the same MHC/peptide determined by X-ray crystallography. The two TCRs differ by three amino acids in the CDR3 α and β loops. As the result different hydrogen bonds are formed between the two CDR3β loops and the peptide in the complexes of the simulated structures, with three hydrogen bonds seen in the TCR-Ob.2F3 complex and five in the TCR-Ob.1A12 complex. The two TCRs, each located near the N-terminal end of the HLA-DR2 binding groove and both had an orthogonal binding axis but they deviated by about 10°. Simulation methods, such as structural docking and molecular dynamics as used here, provide an avenue to understand molecular binding mode efficiently and more rapidly than obtaining multiple crystal structures when a large structural database is already available., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

In T cell-mediated autoimmune diseases, self-reactive T cells with known antigen specificity appear to be particularly promising targets for antigen-specific induction of tolerance without compromising desired protective host immune responses. Several lines of evidence suggest that delivery of antigens to antigen-presenting dendritic cells (DCs) in the steady state (i.e., to immature DCs) may represent a suitable approach to induce antigen-specific T-cell tolerance peripherally. Here, we report that anti-DEC205-mediated delivery of the self-peptide proteolipid protein (PLP)139-151 to DCs ameliorated clinical symptoms in the PLP-induced SJL model of experimental autoimmune encephalomyelitis. Splenocytes from treated mice were anergized to PLP139-151, and IL-17 secretion was markedly reduced. Moreover, we show directly, using transgenic CD4(+) Vβ6(+) TCR T cells specific for PLP139-151, that, under the conditions of the present experiments, these cells also became anergic. In addition, evidence for a CD4(+) T cell-mediated suppressor mechanism was obtained.

Human cytomegalovirus (HCMV) encodes an endoplasmic reticulum (ER)-resident transmembrane glycoprotein, US10, expressed early in the replicative cycle of HCMV as part of the same cluster that encodes the known immunoevasins US2, US3, US6, and US11. We show that US10 down-regulates cell surface expression of HLA-G, but not that of classical class I MHC molecules. The unique and short cytoplasmic tail of HLA-G (RKKSSD) is essential in its role as a US10 substrate, and a tri-leucine motif in the cytoplasmic tail of US10 is responsible for down-regulation of HLA-G. Both the kinetics of HLA-G degradation and the mechanisms responsible appear to be distinct from those used by the US2 and US11 pathways, suggesting the existence of a third route of protein dislocation from the ER. We show that US10-mediated degradation of HLA-G interferes with HLA-G-mediated NK cell inhibition. Given the role of HLA-G in protecting the fetus from attack by the maternal immune system and in directing the differentiation of human dendritic cells to promote the evolution of regulatory T cells, HCMV likely targets the HLA-G-dependent axis of immune recognition no less efficiently than it interferes with classical class I MHC-restricted antigen presentation.

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 WHO Nomenclature Committee for Factors of the HLA System met during the 15th International Histocompatibility and Immunogenetics Workshop in Buzios, Brazil in September 2008. This update is an extract of the main report that documents the additions and revisions to the nomenclature of human leukocyte antigen (HLA) specificities following the principles established in previous reports.

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 approximately 47,400 transcripts. Substantial differences indicate that these two types of NK cells represent distinct subsets., (Copyright 2010 Elsevier Ltd. All rights reserved.)