Rare Cells Session
Wednesday, September 20th, 2023, at 9:00 pm
Chairs: Thomas Kroneis & Frank Schildberg
Continuous technical improvements in the field of single-cell analysis and its data interpretation causes cytometry to find its way into ever more complex problems. This includes, among others, spatial analyzes and analyzes of rare cells or cell populations. In this session we will focus on such analyses, spanning the spectrum from FACS analysis to slide-based spatial biology. In addition to biological questions in connection with rare cells or cell populations, in particular with a focus on microchimerism, there should also be space for contributions dedicated to solving technical difficulties in the analysis of rare events.
Rare cells in pregnancy: the role of regulatory T cells and microchimeric cells in placental and fetal development
Department of Immunology, Leiden University Medical Center, Leiden, The Netherlands
In pregnancies, the mother and the baby are genetically semi-allogeneic to each other, which includes the HLA genotype. This represents an interesting immunologic situation at places where maternal cells have contact with fetal cells. Appropriate development of the placenta is required for healthy pregnancy and delivery of a healthy baby. For this, it is relevant that maternal immunologic tolerance toward fetal cells is maintained. Regulatory T cells (Tregs) constitute up to 5% of the total T cell population and are involved in dampening immune cell activation. Animal models have shown that maternal Tregs are essential for a successful allogeneic pregnancy. Furthermore, women with complicated pregnancy demonstrate decreased Treg numbers at the maternal-fetal interface. In our group, we have been analyzing Treg subsets and effector molecules by spectral flow cytometry both in healthy and complicated human pregnancies. Detailed results are shown and discussed during the lecture.
During gestation, it also has turned out that Tregs from the fetus are relevant in tolerizing maternal allo-antigens. Homing of maternal microchimeric cells in the fetus’ lymphoid organs is a major driver of the development of such tolerogenic fetal Tregs, which persist years after birth. Maternal microchimerism means that cells from the mother transfer over the placenta and end up in the offspring, and it represents a major topic by our Microchimerism, Health and Evolution Consortium formed in 2021. The lecture comprises results concerning frequency and characteristics of these rare microchimeric cells, as obtained by flow cytometric analyses and isolation strategies using HLA monoclonal antibodies.
Michael Eikmans is an immunologist and molecular biologist, and has been performing research in the field of both transplantation and pregnancy. His research group is aimed to understand in healthy pregnancy how the pregnant woman immunologically tolerates her baby, and what mechanisms account for complications to occur in aberrant pregnancy. The mechanisms reflect immune regulatory pathways directed by T cells and macrophages. A considerable part of the work by the group encompasses the study of microchimeric cells and their relevance in pregnancy and transplantation outcomes.
Transplacental migration of maternal natural killer and T cells assessed by ex vivo human placenta perfusion – evidence for microchimerism?
Placenta Laboratory at the University Hospital Jena, Jena, Germany
Introduction: The transplacental passage of cells between a mother and her fetus, known as microchimerism, is a less studied process during pregnancy. The frequency of maternal microchimeric cells in fetal tissues in physiological pregnancies and mechanisms responsible for transplacental cell trafficking are poorly understood. This study aimed to evaluate the placental trafficking of maternal peripheral blood mononuclear cells (PBMC) using human ex vivo placenta perfusion.
Methods: Ten placentas and maternal PBMC were obtained after healthy pregnancies. Flow cytometry was used to characterize PBMC subtypes. The isolated PBMC were stained with a fluorescent dye and perfused through the maternal circuit of the placenta in an ex vivo perfusion system. Subsequent immunofluorescence staining for CD3+ T cells and CD56+ NK cells was performed on placental tissue sections, and the number of detectable PBMC in different tissue areas was counted using fluorescence microscopy.
Results: Peripheral blood showed a higher percentage of CD3+ T cells compared to CD56+ NK cells. Perfused PBMC were detected in all analyzed placentas, with higher numbers in contact with fetal tissue compared to cells without contact. CD3+ T cells were identified more frequently than CD56+ NK cells, and some CD3+ T cells were found inside fetal tissues and vessels. The study also indicates a step-wise mechanism for cell trafficking across the placenta.
Discussion: Maternal PBMC are capable of transmigrating through the syncytiotrophoblast layer into fetal placental tissue and vessels. The findings demonstrate that human placenta perfusion is a suitable method for investigating microchimerism during pregnancy.
Udo Markert is Professor and Head of the Placenta Laboratory at the University Hospital Jena, Germany. He is President of the European Society for Reproductive Immunology (ESRI; 2019-2023) and the current interim Secretary General of the European Placenta Group (EPG; 2020-2023). He has been President of the American Society for Reproductive Immunology (ASRI) 2012-2014, and has served for its Council for 8 years. He has been Councillor also of the International Society for Immunology of Reproduction (ISIR), ESRI and the International Federation of Placenta Societies (IFPA). He has received several awards including the “German Innovation Award Medical Engineering” (2008), the “John Christian Herr Award” of the ASRI (2009) and the American Journal of Reproductive Immunology (AJRI) Award (2016). Udo Markert is Organizer and Chair of the upcoming IFPA congress 2025 in Germany and has organized and co-organized several international conferences, such as the triannual ISIR congress 2016 in Erfurt, and the joint ASRI and ESRI congress 2012 in Hamburg. Since 2022, he is European Editor of “Placenta” and has been European Associate Editor of the AJRI (2010-2021). He is also Editor of several books and special journal issues. He is and was member of further Editorial Boards of other journals in the field. He is visiting professor at the Chongqing Medical University, China.
Udo Markert has studied medicine 1984-1990 at the Heinrich Heine University Düsseldorf, followed by a 2 years internship at its Department of Pathology. 1992 he moved for a post-doctoral training at an Institut National de la Recherche Médical to Paris and came 1996 to Jena. He has worked until 1999 at the Institute of Immunology and since 2000 he has been heading the Placenta Lab at the Department of Obstetrics. He has received his habilitation in 2003 and the professorship in 2013.
Udo Markert’s main research topics are trophoblast and placenta functions, endometrium and ovaries, mostly wth special regard to immunology.
Characterization of Antigen-Specific B Cells and Plasma Cells Using a Tetramer-based Detection Method by Flow and Mass Cytometry
German Rheumatism Research Cente, Berlin, a Leibniz Institute, Berlin, Germany
Numerous studies have demonstrated that antigen-specific serum antibody titers are maintained for many years. Fractions of plasma cells (PC) of the bone marrow persist for very long time and are thus implicated in long-term antibody production. Studying them at the level of antigen-specificity is inevitable to understand the principles of memory induction and maintenance in the B-cell lineage.
We here present an approach for the high-avidity detection of antigen-specific human B cells and PC by flow and mass cytometry based on the combination of biotin-labeled antigens with fluorochrome-/isotope-labelled streptavidin, commonly known as tetramers. The method was validated using human blood and BM cells, and with different protein and viral antigens, including tetanus toxoid, SARS-CoV-2 spike (S1) and receptor binding domain (RBD), epstein barr virus EBNA1, and monkeypox virus antigens. Assay specificity was validated using biological controls, e.g., by reproducing expected cellular dynamics in response to vaccination. The approach was compatible with live and fixed cells, cell-surface and intracellular staining. In multiplexed analyses, up to four antigen specificities were assessed in the same assay, using combinatorial tetramer labeling.
Using SARS-CoV-2 RBD- and S1- tetramers, we identified phenotypically distinct subsets of SARS‑CoV-2-specific PC within the human bone marrow of 16 donors after basic mRNA immunization. We found SARS-CoV-2-S1-specific PC, representing 0.22% of total BMPC, the majority expressing IgG, which indicates their emergence from a systemic vaccination response. Notably, one-fifth of SARS-CoV-2-specific PC showed the phenotype of long-lived memory plasma cells characterized by downregulated CD19 and present or absent CD45 expression.
Taken together, we established multiplexed detection of various antigen-specific B cell subsets in a single assay, providing analytical access to B cell responses at single-cell levels from limited sample size in infection, vaccination, and autoimmunity.
Image-based SNP detection for discriminating haploidentical microchimeric cells
Medical University of Graz; Gottfried Schatz Research Center for Cell Signalling, Metabolism and Aging; Division of Cell Biology, Histology and Embryology, Graz, Austria
Pregnancy associated microchimerism research in humans is historically dominated by PCR based approaches targeting polymorphisms in the DNA. However, while those methods allow for sensitive detection of microchimeric sequences in various DNA extracts, most information on cell type, spatial localization and cellular activity is lost. It would be fair to say that the lack of a suitable method to identify and characterize maternal and fetal microchimeric cells within their spatial context, has obstructed significant progress over the last decade. Here we present or ongoing work on developing an imaging based, in situ approach to discriminate between haploidentical cells. We employ rolling circle amplification of padlock probes targeting InDels and SNPs present in the mRNA, to generate discrete point signals within a cell. This will allow us to visualize, in a sex-unbiased manner, microchimeric cells within their native tissues.