BackgroundDuring mammalian pregnancy, a maternal immunoregulatory network develops in the decidua that fosters fetal development, maintains tolerance to fetal antigens and protects against infection. Herein, decidual regulatory T cells (Tregs) are crucial for pregnancy success.Aim and methodologyTo understand the nature and response of maternal CD4+ Tregs and CD4+ conventional T-cells (Tconv) in healthy human pregnancy, we analyzed these cells in decidua and blood at term, caesarean delivery by single-cell transcriptomics and TCR sequencing.ResultsData mining revealed novel discerning, as well as shared features and functionalities of decidual CD4+ Tregs and Tconvs. Both cell types showed evidence of antigen recognition and activation in the decidua, followed by local clonal expansion and effector differentiation. Tregs were largely of the thymus-derived (t)Treg lineage that has a self-antigen reactive T cell receptor (TCR) repertoire. Tregs locally expanded, more so than Tconvs, and differentiated into typical non-lymphoid tissue (NLT)-resident effector cells with discerning cell surface markers and multiple suppressive functions, driven by TNF receptor-2 costimulation. Additional factors, including IFNs, interleukins and prolactin emerged as shared drivers of decidual Treg and Tconv effector differentiation. Effector Tconvs had cell lineage-discerning proinflammatory and cytotoxic capacities, restrained by cell-intrinsic and -extrinsic mechanisms, while sharing glycolytic and antigen-presenting features with Tregs. Treg and Tconv subpopulations showed signs of exhaustion, suggesting chronic antigenic stimulation. Overall, the observed features argue for ongoing de novo T-cell priming and dynamic T-cell turnover in the decidua throughout pregnancy.ConclusionOur findings suggest that non-self-reactive CD4+ Tconvs and self-reactive tTregs are continuously primed in lymphoid organs during pregnancy, reactivated by antigen in the decidua and dynamically turned over. In the decidua, CD4+ Tconvs and Tregs expand and differentiate under influence of specific cytokines into tissue-resident effector cells with opposing and restrained pro- and anti-inflammatory functions, as well as shared antigen-presenting capacity.SignificanceThese molecular definitions of decidual CD4+ Tregs and Tconvs can be used to aid diagnostics at mRNA and protein level. Moreover, they facilitate mechanistic understanding of maternal-fetal tolerance, based on extrapolation of known T-cell lineage characteristics and tissue adaptations in health and disease.

OBJECTIVE:Rheumatoid arthritis (RA) is characterized by anti-modified protein antibodies (AMPAs), including anti-citrullinated protein antibodies (ACPA), anti-carbamylated protein antibodies (anti-CarP), and anti-acetylated protein antibodies (AAPA). In contrast to other AMPAs, AAPA IgM is found in healthy individuals, raising questions about its role in early immune responses. We investigated whether AAPA IgM serves as a precursor for other AMPAs, marking the initial breach of tolerance in RA. METHODS:AAPA IgM levels were measured in cord blood and serum of children up to three years old to assess whether it represents a natural (auto)antibody. To evaluate whether AAPA IgM presence precedes other AMPAs, we longitudinally measured AAPA and ACPA IgM in individuals before RA onset. To assess whether AAPA IgM-expressing B cells display a naive phenotype and carry germline-encoded B cell receptors (BCRs), single cells were sorted and sequenced. RESULTS:AAPA IgM was not detected in early life, but before RA onset, significantly more individuals were AAPA IgM positive (27.3%) compared to ACPA IgM positive (11.7%). Toward disease onset, ACPA IgM positivity increased to 51.2%, whereas AAPA IgM positivity remained stable. Furthermore, AMPAs developed in individuals who were AAPA IgM negative. Regarding B cell characteristics, germline-encoded BCRs were identified among both AAPA- and ACPA-expressing B cells in patients with RA. CONCLUSION:AMPA responses in RA do not appear to arise solely from AAPA IgM, as suggested by the lack of association between predisease AMPA IgM positivity and later AMPA evolution. This is further supported by the presence of germline-configured ACPA BCRs, suggesting multiple possible starting points for AMPA responses.

Microchimerism is defined as the presence of a small population of genetically distinct cells within a host that is derived from another individual. Throughout pregnancy, maternal and fetal cells are known to traffic across the feto-maternal interface and result in maternal and fetal microchimerism, respectively. However, the routes of cell transfer, the molecular signaling as well as the timing in which trafficking takes place are still not completely understood. Recently, the presence of inflammation at the feto-maternal interface has been linked with maternal microchimeric cells modulating organ development in the fetus. Here, we review the current literature and suggest that inflammatory processes at the feto-maternal interface tissues are a physiological prerequisite for the establishment of microchimerism. We further propose a spatio-temporal corridor of microchimeric cell migration to potentially explain some biological effects of microchimerism. Additionally, we elaborate on the possible consequences of a shift in this spatio-temporal corridor, potentially responsible for the development of pathologies in the neonate.

Microchimerism research has recently gained renewed attention despite known existence of these rare cells for decades. Fetal and maternal microchimeric-derived cells may have functional capabilities, and are increasingly associated with both beneficial and adverse health outcomes. Yet, establishing the role of microchimerism in health has been largely constrained methodologically and theoretically. The Microchimerism, Human Health, and Evolution Project address these challenges by calling on 29 leading microchimerism experts to put forth key research questions that can substantially advance the field. Seven major categories are identified: function and mechanism; microchimerism in interventions, treatment, and transplant; mapping "generational microchimerism"; evolution; microchimerism detection; appropriate experimental model systems; and definition of microchimerism. Identifying these questions - and continuing to find answers - will be crucial for advancing the knowledge of microchimerism in health and disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the placenta can lead to fetal distress and demise, characterized by severe trophoblast necrosis, chronic histiocytic intervillositis (CHI), and massive perivillous fibrin deposition. We aimed to uncover spatial immune-related protein changes in SARS-CoV-2 placentitis compared with CHI placentas and uncomplicated pregnancies to gain insight into the underlying pathophysiological mechanisms. Placentas were retrospectively collected from cases with SARS-CoV-2 placentitis resulting in fetal distress/demise (n = 9), CHI (n = 9), and uncomplicated term controls (n = 9). The expression of 53 immune-related proteins was quantified using GeoMx Digital Spatial Profiler in three separate compartments: villi (fetal compartment), intervillous space, and decidua (both maternal compartments). Compared with controls, SARS-CoV-2 placentitis and CHI both displayed differentially expressed proteins in the intervillous space only, including upregulation of myeloid markers (e.g., CD40, CD11c, CD68, CD163). Specifically, SARS-CoV-2 placentitis was associated with reduced expression of multiple apoptotic proteins (e.g., BAD, BIM, BLXL, BCL6). In conclusion, SARS-CoV-2 placentitis and CHI are associated with enhanced myeloid cell infiltration into the intervillous space, but not in the decidua and villi. The more prominently reduced apoptosis-related protein expression in SARS-CoV-2 placentitis may lead to an exaggerated immune response, causing acute placental dysfunction and fetal demise.

IntroductionMaternal-fetal HLA compatibility influences pregnancy outcome, including preeclampsia risk. Cervical extravillous trophoblasts (EVT) in early pregnancy provide a non-invasive source for fetal genome acquisition, potentially enabling fetal HLA typing for obstetric risk assessment. This study aimed to achieve fetal HLA typing through EVT isolation using HLA-G-coupled nanoparticle immunomagnetic separation (TRIC) and fluorescence-activated cell sorting (FACS).MethodCervical samples from 32 pregnant women were collected by cytobrush. Saliva and umbilical cord blood (n=13) served as maternal and fetal HLA genotype controls, respectively. Cervical samples from non-pregnant women, primary cultured EVT, and cryo-sectioned term placentas served as controls for cell phenotype, protein expression, and effect of fixation. FACS and TRIC were applied to isolate EVT from maternal cells, followed by RSSO-PCR for HLA typing. EVT presence pre- and post-isolation was determined through HLA-G, β-hCG, and Cytokeratin-7 (CK-7) expression. TRIC was optimized by improving antibody-binding-efficiency, and comparing three (nano)beads types and two magnets.ResultsPurity and yield of HLA-G+β-hCG+CK-7+ cells after TRIC failed to match pre-isolation HLA-G+ cell counts, despite protocol optimization. FACS revealed a fetal HLA genotype. In contrast, only the maternal HLA genotype was detected in TRIC-isolated cells.ConclusionEVT counts and maternal cell contamination limit reliable fetal HLA typing from cervical samples. Refining non-invasive EVT isolation techniques may enable fetal HLA typing to be included in risk assessment of pregnancy complications.

BACKGROUND:Despite almost 3 decades of research, the mechanisms underlying the bidirectional trafficking of cells at the maternal-fetal interface that gives rise to microchimerism remain poorly understood. A major barrier to progress has been the lack of suitable detection methods capable of distinguishing maternal from fetal cells within the spatial context of the human placenta. To address this, we developed a novel detection method based on padlock probe technology to differentiate haploidentical cells in placental tissues. METHODS:Padlock probes were designed to target single nucleotide polymorphisms (SNPs) present in messenger RNA transcripts. The assays were first validated in cell lines and subsequently applied to placental tissue to assess its ability to distinguish between maternal and fetal cells. RESULTS:We established a panel of 27 assays targeting 3 human leukocyte antigen-A alleles and 12 biallelic SNPs. The method demonstrated high specificity and sensitivity, detecting minor cell populations at dilutions as low as 1:10 000. Proof of concept was obtained in a decidua basalis specimen, showing the assays' capability to distinguish maternal and fetal cells within placental tissue. CONCLUSIONS:We present a novel, sex-unbiased methodology for the in situ visualization of haploidentical (microchimeric) cells. This approach enables the study of maternal-fetal cellular interactions within their native tissues at the maternal-fetal interface.

Introduction Recurrent pregnancy loss (RPL) is defined as the occurrence of two or more spontaneous pregnancy losses from the time of conception until 24 weeks of gestation. Currently, an underlying cause can be identified in only a minority of the losses. Potentially, an impaired maternal immune response targeting the semiallograft pregnancy may lead to miscarriage. While prior studies have explored the use of immune-suppressing corticosteroids to modulate the maternal immune system and hopefully improve pregnancy outcome, the absence of sufficiently powered randomised controlled trials (RCT) underscores the need for further research. The primary aim of this study is to evaluate if prednisolone administration in early pregnancy (20 mg daily for 6 weeks, then tapering doses for 2 weeks) in women with unexplained RPL leads to a higher live birth rate (LBR) in comparison to placebo. Additionally, the study assesses the tolerability, safety and the cost-effectiveness of this intervention. Finally, we will explore the effect of prednisolone in various subgroups (based on maternal age, number of previous pregnancy losses, presence of specific antibodies and pre-pregnancy endometrial immune cell level).Methods and analysis This ongoing multicentre, double-blind RCT will randomise 490 women with unexplained RPL and pregnancy <7 weeks to receive either prednisolone or placebo. Each participant will be followed up for 1 year, with digital questionnaires to assess depression, anxiety, medical expenses and productivity loss. We will also collect data on maternal and paternal demographics and neonatal outcomes. The sample size of 490 participants was calculated according to a minimally important increase in LBR of 12% (expecting a LBR of 63% in the general RPL population), including loss to follow-up (estimated at 5%). The analysis will follow the intention-to-treat principle.Ethics and dissemination This study was submitted under the Clinical Trial Regulation (CTR) in Clinical Trials Information System (CTIS) for assessment by the Central Committee on Research Involving Human Subjects (CCMO) under Clinical Trial number: 2023-503220-76-01. It received full approval on 29/01/2024. Study findings will be presented at conferences and published in a peer-reviewed journal. Participants will be informed about the results by publishing them on the publicly available website of the study.Trial registration number This trial is registered in ClinicalTrials.gov (ID NCT05725512) and in CTIS (2023-503220-76-01).