CD4+ T cell heterogeneity in gestational age and preeclampsia using single-cell RNA sequencing.

A balance between pro-inflammatory decidual CD4+ T cells and FOXP3+ regulatory T cells (FOXP3+ Tregs) is important for maintaining fetomaternal tolerance. Using single-cell RNA-sequencing and T cell receptor repertoire analysis, we determined that diversity and clonality of decidual CD4+ T cell subsets depend on gestational age. Th1/Th2 intermediate and Th1 subsets of CD4+ T cells were clonally expanded in both early and late gestation, whereas FOXP3+ Tregs were clonally expanded in late gestation. Th1/Th2 intermediate and FOXP3+ Treg subsets showed altered gene expression in preeclampsia (PE) compared to healthy late gestation. The Th1/Th2 intermediate subset exhibited elevated levels of cytotoxicity-related gene expression in PE. Moreover, increased Treg exhaustion was observed in the PE group, and FOXP3+ Treg subcluster analysis revealed that the effector Treg like subset drove the Treg exhaustion signatures in PE. The Th1/Th2 intermediate and effector Treg like subsets are possible inflammation-driving subsets in PE.

Splicing neoantigen discovery with SNAF reveals shared targets for cancer immunotherapy.

Immunotherapy has emerged as a crucial strategy to combat cancer by "reprogramming" a patient's own immune system. Although immunotherapy is typically reserved for patients with a high mutational burden, neoantigens produced from posttranscriptional regulation may provide an untapped reservoir of common immunogenic targets for new targeted therapies. To comprehensively define tumor-specific and likely immunogenic neoantigens from patient RNA-Seq, we developed Splicing Neo Antigen Finder (SNAF), an easy-to-use and open-source computational workflow to predict splicing-derived immunogenic MHC-bound peptides (T cell antigen) and unannotated transmembrane proteins with altered extracellular epitopes (B cell antigen). This workflow uses a highly accurate deep learning strategy for immunogenicity prediction (DeepImmuno) in conjunction with new algorithms to rank the tumor specificity of neoantigens (BayesTS) and to predict regulators of mis-splicing (RNA-SPRINT). T cell antigens from SNAF were frequently evidenced as HLA-presented peptides from mass spectrometry (MS) and predict response to immunotherapy in melanoma. Splicing neoantigen burden was attributed to coordinated splicing factor dysregulation. Shared splicing neoantigens were found in up to 90% of patients with melanoma, correlated to overall survival in multiple cancer cohorts, induced T cell reactivity, and were characterized by distinct cells of origin and amino acid preferences. In addition to T cell neoantigens, our B cell focused pipeline (SNAF-B) identified a new class of tumor-specific extracellular neoepitopes, which we termed ExNeoEpitopes. ExNeoEpitope full-length mRNA predictions were tumor specific and were validated using long-read isoform sequencing and in vitro transmembrane localization assays. Therefore, our systematic identification of splicing neoantigens revealed potential shared targets for therapy in heterogeneous cancers.

Antigen-specific decidual CD8+ T cells include distinct effector memory and tissue-resident memory cells.

Maternal decidual CD8+ T cells must integrate the antithetical demands of providing immunity to infection while maintaining immune tolerance for fetal and placental antigens. Human decidual CD8+ T cells were shown to be highly differentiated memory T cells with mixed signatures of dysfunction, activation, and effector function. However, no information is present on how specificity for microbial or fetal antigens relates to their function or dysfunction. In addition, a key question, whether decidual CD8+ T cells include unique tissue-resident memory T cells (Trm) or also effector memory T cell (Tem) types shared with peripheral blood populations, is unknown. Here, high-dimensional flow cytometry of decidual and blood CD8+ T cells identified 2 Tem populations shared in blood and decidua and 9 functionally distinct Trm clusters uniquely found in decidua. Interestingly, fetus- and virus-specific decidual CD8+ Trm cells had similar features of inhibition and cytotoxicity, with no significant differences in their expression of activation, inhibitory, and cytotoxic molecules, suggesting that not all fetus-specific CD8+ T cell responses are suppressed at the maternal-fetal interface. Understanding how decidual CD8+ T cell specificity relates to their function and tissue residency is crucial in advancing understanding of their contribution to placental inflammation and control of congenital infections.

Purification of primary human placental leukocytes to study maternal-fetal interactions.

Decidual leukocytes play key roles in maternal-fetal tolerance and immunity. Here, we present detailed methods to purify, culture, and functionally analyze human placental dNK, dTreg, dTem, and dMɸ from decidua parietalis, the maternal part of the placental membranes; decidua basalis, the maternal part of the placenta; and placental villi. These sites have high clinical relevance in the development of villitis and chorioamnionitis. This allows in-depth phenotypic and functional investigation of placental immune populations and their interactions with extravillous trophoblasts. For complete details on the use and execution of this protocol, please refer to Ikumi et al.,1 Tilburgs et al.,2 Salvany-Celades et al.,3 Crespo et al.,4 van der Zwan et al.5.

Utilizing primary HLA-G+ extravillous trophoblasts and HLA-G+ EVT-like cell lines to study maternal-fetal interactions.

Fetal extravillous trophoblasts (EVTs) are the most invasive cells of the placenta and play a key role in modulating maternal immune responses. Here, we present a protocol to purify and culture human leukocyte antigen-G (HLA-G)+ EVTs. We describe steps for tissue dissection, tissue digestion, density gradient centrifugation, and cell sorting, and we provide detailed methods to determine EVT function. HLA-G+ EVTs are isolated from two maternal-fetal interfaces: the chorionic membrane and the basalis/villous tissue. This protocol allows in-depth functional investigation of maternal immune interactions with HLA-G+ EVTs. For complete details on the use and execution of this protocol, please refer to Papuchova et al. (2020),1 Salvany-Celades et al. (2019),2 Tilburgs et al. (2015),3 Tilburgs et al. (2015),4 van der Zwan et al. (2018).5.

BAFF and APRIL counterregulate susceptibility to inflammation-induced preterm birth.

Clinical evidence points to a function for B cell-activating factor (BAFF) in pregnancy. However, direct roles for BAFF-axis members in pregnancy have not been examined. Here, via utility of genetically modified mice, we report that BAFF promotes inflammatory responsiveness and increases susceptibility to inflammation-induced preterm birth (PTB). In contrast, we show that the closely related A proliferation-inducing ligand (APRIL) decreases inflammatory responsiveness and susceptibility to PTB. Known BAFF-axis receptors serve a redundant function in signaling BAFF/APRIL presence in pregnancy. Treatment with anti-BAFF/APRIL monoclonal antibodies or BAFF/APRIL recombinant proteins is sufficient to manipulate susceptibility to PTB. Notably, macrophages at the maternal-fetal interface produce BAFF, while BAFF and APRIL presence divergently shape macrophage gene expression and inflammatory function. Overall, our findings demonstrate that BAFF and APRIL play divergent inflammatory roles in pregnancy and provide therapeutic targets for mitigating risk of inflammation-induced PTB.

Maternal-fetal conflict averted by progesterone- induced FOXP3+ regulatory T cells.

Pregnancy stimulates an intricately coordinated assortment of physiological changes to accommodate growth of the developing fetus, while simultaneously averting rejection of genetically foreign fetal cells and tissues. Despite increasing evidence that expansion of immune-suppressive maternal regulatory T cells enforces fetal tolerance and protects against pregnancy complications, the pregnancy-associated signals driving this essential adaptation remain poorly understood. Here we show that the female reproductive hormone, progesterone, coordinates immune tolerance by stimulating expansion of FOXP3+ regulatory T cells. Conditional loss of the canonical nuclear progesterone receptor in maternal FOXP3+ regulatory T cells blunts their proliferation and accumulation, which is associated with fetal wastage and decidual infiltration of activated CD8+ T cells. Reciprocally, the synthetic progestin 17α-hydroxyprogesterone caproate (17-OHPC) administered to pregnant mice reinforces fetal tolerance and protects against fetal wastage. These immune modulatory effects of progesterone that promote fetal tolerance establish a molecular link between immunological and other physiological adaptions during pregnancy.

Purification of Primary Decidual Natural Killer Cells for Functional Analysis.

Decidual NK cells (dNK) are a unique type of NK cells found at the maternal-fetal interface during pregnancy. dNK play a key role in placental development, trophoblast invasion, and immunity to viral and bacterial infection of the placenta. dNK are the predominant leukocyte population in first trimester placental tissues and comprise around 70% of the total CD45+ leukocytes. dNK remain present throughout pregnancy but their proportion decreases to 20-40% of term placenta decidual tissue leukocytes. Investigation of dNK function throughout pregnancy is of high clinical relevance for understanding the development of placental inflammatory disorders as well as maternal-to-fetal transmission of pathogens. In this chapter, we describe in detail the methods we developed to purify dNK from first trimester and term pregnancy placental tissues. These methods are suitable to assess their protein and gene expression profiles as well as their function.

Dysregulated non-coding telomerase RNA component and associated exonuclease XRN1 in leucocytes from women developing preeclampsia-possible link to enhanced senescence.

Senescence in placenta/fetal membranes is a normal phenomenon linked to term parturition. However, excessive senescence which may be induced by telomere attrition, has been associated with preeclampsia (PE). We hypothesized that the telomerase complex in peripheral blood mononuclear cells (PBMC) and circulating telomere associated senescence markers would be dysregulated in women with PE. We measured long non-coding (nc) RNA telomerase RNA component (TERC) and RNAs involved in the maturation of TERC in PBMC, and the expression of TERC and 5'-3' Exoribonuclease 1 (XRN1) in extracellular vesicles at 22-24 weeks, 36-38 weeks and, 5-year follow-up in controls and PE. We also measured telomere length at 22-24 weeks and 5-year follow-up. The circulating senescence markers cathelicidin antimicrobial peptide (CAMP), ß-galactosidase, stathmin 1 (STMN1) and chitotriosidase/CHIT1 were measured at 14-16, 22-24, 36-38 weeks and at 5-year follow-up in the STORK study and before delivery and 6 months post-partum in the ACUTE PE study. We found decreased expression of TERC in PBMC early in pregnant women who subsequently developed PE. XRN1 involved in the maturation of TERC was also reduced in pregnancy and 5-year follow-up. Further, we found that the senescence markers CAMP and ß-galactosidase were increased in PE pregnancies, and CAMP remained higher at 5-year follow-up. ß-galactosidase was associated with atherogenic lipid ratios during pregnancy and at 5-year follow-up, in PE particularly. This study suggests a potential involvement of dysfunctional telomerase biology in the pathophysiology of PE, which is not restricted to the placenta.

T-Cell Homeostatic Imbalance in Placentas From Women With Human Immunodeficiency Virus in the Absence of Vertical Transmission.

BACKGROUND: Implementation of universal antiretroviral therapy (ART) has significantly lowered vertical transmission rates but has also increased numbers of human immunodeficiency virus (HIV)-exposed uninfected children, who remain vulnerable to morbid effects. In the current study, we investigated whether T-cell alterations in the placenta contribute to altered immune status in HIV-exposed uninfected. METHODS: We analyzed T cells from term placenta decidua and villous tissue and paired cord blood from pregnant women living with HIV (PWH) who initiated ART late in pregnancy (n = 21) with pregnant women not living with HIV (PWNH) (n = 9). RESULTS: Placentas from PWH showed inverted CD4/CD8 ratios and higher proportions of tissue resident CD8+ T cells in villous tissue relative to control placentas. CD8+ T cells in the fetal capillaries, which were of fetal origin, were positively correlated with maternal plasma viremia before ART initiation, implying that imbalanced T cells persisted throughout pregnancy. In addition, the expanded memory differentiation of CD8+ T cells was confined to the fetal placental compartment and cord blood but was not observed in the maternal decidua. CONCLUSIONS: T-cell homeostatic imbalance in the blood circulation of PWH is reflected in the placenta. The placenta may be a causal link between HIV-induced maternal immune changes during gestation and altered immunity in newborn infants in the absence of vertical transmission.

ELF3 activated by a superenhancer and an autoregulatory feedback loop is required for high-level HLA-C expression on extravillous trophoblasts.

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.

Maternal regulation of inflammatory cues is required for induction of preterm birth.

Infection-driven inflammation in pregnancy is a major cause of spontaneous preterm birth (PTB). Both systemic infection and bacterial ascension through the vagina/cervix to the amniotic cavity are strongly associated with PTB. However, the contribution of maternal or fetal inflammatory responses in the context of systemic or localized models of infection-driven PTB is not well defined. Here, using intraperitoneal or intraamniotic LPS challenge, we examined the necessity and sufficiency of maternal and fetal Toll-like receptor (TLR) 4 signaling in induction of inflammatory vigor and PTB. Both systemic and local LPS challenge promoted induction of inflammatory pathways in uteroplacental tissues and induced PTB. Restriction of TLR4 expression to the maternal compartment was sufficient for induction of LPS-driven PTB in either systemic or intraamniotic challenge models. In contrast, restriction of TLR4 expression to the fetal compartment failed to induce LPS-driven PTB. Vav1-Cre-mediated genetic deletion of TLR4 suggested a critical role for maternal immune cells in inflammation-driven PTB. Further, passive transfer of WT in vitro-derived macrophages and dendritic cells to TLR4-null gravid females was sufficient to induce an inflammatory response and drive PTB. Cumulatively, these findings highlight the critical role for maternal regulation of inflammatory cues in induction of inflammation-driven parturition.

Decidual NK Cells Transfer Granulysin to Selectively Kill Bacteria in Trophoblasts.

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.

Endometrial Decidualization: The Primary Driver of Pregnancy Health.

Interventions to prevent pregnancy complications have been largely unsuccessful. We suggest this is because the foundation for a healthy pregnancy is laid prior to the establishment of the pregnancy at the time of endometrial decidualization. Humans are one of only a few mammalian viviparous species in which decidualization begins during the latter half of each menstrual cycle and is therefore independent of the conceptus. Failure to adequately prepare (decidualize) the endometrium hormonally, biochemically, and immunologically in anticipation of the approaching blastocyst-including the downregulation of genes involved in the pro- inflammatory response and resisting tissue invasion along with the increased expression of genes that promote angiogenesis, foster immune tolerance, and facilitate tissue invasion-leads to abnormal implantation/placentation and ultimately to adverse pregnancy outcome. We hypothesize, therefore, that the primary driver of pregnancy health is the quality of the soil, not the seed.

Three types of HLA-G+ extravillous trophoblasts that have distinct immune regulatory properties.

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.

Human Term Pregnancy Decidual NK Cells Generate Distinct Cytotoxic Responses.

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.

Three Types of Functional Regulatory T Cells Control T Cell Responses at the Human Maternal-Fetal Interface.

During pregnancy, maternal regulatory T cells (Tregs) are important in establishing immune tolerance to invading fetal extravillous trophoblasts (EVTs). CD25HIFOXP3+ 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: CD25HIFOXP3+, PD1HIIL-10+, and TIGIT+FOXP3dim. Moreover, co-culture of HLA-G+ EVTs or decidual macrophages with blood CD4+ T cells directly increased the proportions of CD25HIFOXP3+ Tregs compared to T cells cultured alone. EVTs also increased PD1HI 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.

The Dual Role of HLA-C in Tolerance and Immunity at the Maternal-Fetal Interface.

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.

Mixed signature of activation and dysfunction allows human decidual CD8+ T cells to provide both tolerance and immunity.

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.