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.

Human HLA-G+ extravillous trophoblasts: Immune-activating cells that interact with decidual leukocytes.

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.

Screening assay for blood vessel maturation inhibitors.

In cancer patients, the development of resistance to anti-angiogenic agents targeting the VEGF pathway is common. Increased pericyte coverage of the tumor vasculature undergoing VEGF targeted therapy has been suggested to play an important role in resistance. Therefore, reducing the pericytes coverage of the tumor vasculature has been suggested to be a therapeutic approach in breaking the resistance to and increasing the efficacy of anti-angiogenic therapies. To screen compound libraries, a simple in vitro assay of blood vessel maturation demonstrating endothelial cells and pericytes association while forming lumenized vascular structures is needed. Unfortunately, previously described 3-dimensional, matrix based assays are laborious and challenging from an image and data acquisition perspective. For these reasons they generally lack the scalability needed to perform in a high-throughput environment. With this work, we have developed a novel in vitro blood vessel maturation assay, in which lumenized, vascular structures form in one optical plane and mesenchymal progenitor cells (10T1/2) differentiate into pericyte-like cells, which associate with the endothelial vessels (HUVECs). The differentiation of the 10T1/2 cells into pericyte-like cells is visualized using a GFP reporter controlled by the alpha smooth muscle actin promoter (SMP-8). The organization of these vascular structures and their recruited mural cells in one optical plane allows for automated data capture and subsequent image analysis. The ability of this assay to screen for inhibitors of pericytes recruitment was validated. In summary, this novel assay of in vitro blood vessel maturation provides a valuable tool to screen for new agents with therapeutic potential.

Imaging mass cytometry reveals the prominent role of myeloid cells at the maternal-fetal interface.

Although the immunological complexity of the maternal-fetal interface is well appreciated, the actual interaction of maternal immune cells and fetal trophoblasts is insufficiently understood. To comprehend the composition and spatial orientation of maternal immune cells and fetal extravillous trophoblasts, we applied imaging mass cytometry on decidua basalis of the three trimesters of healthy pregnancy. Within all trimesters, we observed considerably higher frequencies of myeloid cells in the decidua than is seen with single-cell suspension techniques. Moreover, they were the most pronounced cell type in the microenvironment of other decidual cells. In first trimester, HLA-DR- macrophages represented the most abundant myeloid subcluster and these cells were frequently observed in the vicinity of trophoblasts. At term, HLA-DR+ macrophage subclusters were abundantly present and frequently observed in the microenvironment of T cells. Taken together, our results highlight the dynamic role of myeloid cells at the human maternal-fetal interface throughout gestation.

Got your mother in a whirl: The role of maternal T cells and myeloid cells in pregnancy.

Appropriate development of the placenta is required for healthy pregnancy to occur. After implantation of the fertilized blastocyst, fetal trophoblasts invade the endometrium and myometrium of the mother's uterus to establish placentation. In this process, fetal trophoblasts encounter maternal immune cells. In this review, we focus on the role of maternal T cells and myeloid cells (macrophages, dendritic cells) in pregnancy and their interaction with trophoblasts. To retain immunologic tolerization, trophoblasts evade immune recognition by T cells and produce factors that modulate their phenotype and function. On top of that, the local environment at the maternal-fetal interface favors expansion of regulatory T cells. Macrophages and dendritic cells are essential in maintaining a healthy pregnancy. They produce soluble factors and act as antigen-presenting cells, thereby interacting with T cells. Herein, M2 macrophages, immature dendritic cells, CD4+ Th2 cells, and regulatory T cells represent an axis that maintains a local immune tolerant environment. We consider outstanding issues concerning these cell types and their pathways, which need to be addressed in future investigations. Data from recent single-cell sequencing experiments of the placental bed, to study heterogeneity of maternal immune cells and to predict cell-cell interactions, are discussed. Novel ways for long-term culturing of primary trophoblasts allow for cell-cell interaction studies in a functional way. Future directions should include study of the functionality of currently known and newly identified decidual immune cell subsets in healthy and complicated pregnancies, and their interaction with and modulation by trophoblast cells.

Visualizing Dynamic Changes at the Maternal-Fetal Interface Throughout Human Pregnancy by Mass Cytometry.

During healthy pregnancy, a balanced microenvironment at the maternal-fetal interface with coordinated interaction between various immune cells is necessary to maintain immunological tolerance. While specific decidual immune cell subsets have been investigated, a system-wide unbiased approach is lacking. Here, mass cytometry was applied for data-driven, in-depth immune profiling of the total leukocyte population isolated from first, second, and third trimester decidua, as well as maternal peripheral blood at time of delivery. The maternal-fetal interface showed a unique composition of immune cells, different from peripheral blood, with significant differences between early and term pregnancy samples. Profiling revealed substantial heterogeneity in the decidual lymphoid and myeloid cell lineages that shape gestational-specific immune networks and putative differentiation trajectories over time during gestation. Uncovering the overall complexity at the maternal-fetal interface throughout pregnancy resulted in a human atlas that may serve as a foundation upon which comprehension of the immune microenvironment and alterations thereof in pregnancy complications can be built.

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.

Cross-Reactivity of Virus-Specific CD8+ T Cells Against Allogeneic HLA-C: Possible Implications for Pregnancy Outcome.

Heterologous immunity of virus-specific T cells poses a potential barrier to transplantation tolerance. Cross-reactivity to HLA-A and -B molecules has broadly been described, whereas responses to allo-HLA-C have remained ill defined. In contrast to the transplant setting, HLA-C is the only polymorphic HLA molecule expressed by extravillous trophoblasts at the maternal-fetal interface during pregnancy. Uncontrolled placental viral infections, accompanied by a pro-inflammatory milieu, can alter the activation status and stability of effector T cells. Potential cross-reactivity of maternal decidual virus-specific T cells to fetal allo-HLA-C may thereby have detrimental consequences for the success of pregnancy. To explore the presence of cross-reactivity to HLA-C and the other non-classical HLA antigens expressed by trophoblasts, HLA-A and -B-restricted CD8+ T cells specific for Epstein-Barr virus, Cytomegalovirus, Varicella-Zoster virus, and Influenza virus were tested against target cells expressing HLA-C, -E, and -G molecules. An HLA-B*08:01-restricted EBV-specific T cell clone displayed cross-reactivity against HLA-C*01:02. Furthermore, cross-reactivity of HLA-C-restricted virus-specific CD8+ T cells was observed for HCMV HLA-C*06:02/TRA CD8+ T cell lines and clones against HLA-C*03:02. Collectively, these results demonstrate that cross-reactivity against HLA-C can occur and thereby may affect pregnancy outcome.

Cytotoxic potential of decidual NK cells and CD8+ T cells awakened by infections.

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.