Adaptation and Modulation of Memory and Regulatory T Cells in Pregnancy
Kieffer, Tom Eduard Christiaan
DOI:
10.33612/diss.97355536
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Kieffer, T. E. C. (2019). Adaptation and Modulation of Memory and Regulatory T Cells in Pregnancy. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.97355536
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Regulatory T Cells in Pregnancy
Author: Tom E.C. Kieffer
Cover design and artwork: Lisanne Koopmans | Kijk om te Zien Text lay-out: Sandra Tukker | Ridderprint Print: Ridderprint | www.ridderprint.nl ISBN (printed book): 978-94-034-1901-5
ISBN (electronic book): 978-94-034-1900-8 This PhD project was financially supported by: University Medical Center Groningen University of Groningen
Junior Scientific Masterclass, Faculty of Medicine, University of Groningen Jan Kornelis de Cock Foundation
Fonds Gezond Geboren J.C. Ruigrok Stichting
Research Foundation Obstetrics and Gynaecology, University Medical Center Groningen Copyright © 2019 by Tom Kieffer
All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means without permission of the copyright owner.
of Memory and Regulatory
T Cells in Pregnancy
Proefschrift
ter verkrijging van de graad van doctor aan de
Rijksuniversiteit Groningen
op gezag van de
rector magnificus prof. dr. C. Wijmenga
en volgens besluit van het College voor Promoties.
De openbare verdediging zal plaatsvinden op
woensdag 25 september 2019 om 16.15 uur
door
Tom Eduard Christiaan Kieffer
geboren op 17 augustus 1991
Copromotores Dr. M.M. Faas Dr. J.R. Prins Beoordelingscommissie Prof. dr. F.H.J. Claas Prof. dr. J.J.H.M. Erwich Prof. dr. F.G.M. Kroese
1. General Introduction 11
2. Review: Memory T Cells in Pregnancy 27
Kieffer T.E.C., Laskewitz A., Scherjon S.A., Faas M.M., Prins J.R. Frontiers in Immunology 2019; 10:624
Part 1 Physiology: Adaptations of Memory and Regulatory 63
T Cells in Uncomplicated Pregnancies
3. Pregnancy persistently affects memory T cell populations 65
Kieffer T.E.C., Faas M.M., Scherjon S.A., Prins J.R. Journal of Reproductive Immunology 2017; 119:1–8
4. A Different Immune Phenotype in Decidual Tissue from Multigravid 85 Women Compared to Primigravid Women
Kieffer T.E.C., Laskewitz A., Erwich J.J.H.M., Scherjon S.A., Faas M.M., Prins J.R. (Manuscript in preparation)
5. Lower FOXP3 mRNA expression in first trimester decidual tissue 105
from uncomplicated term pregnancies with a male fetus
Kieffer T.E.C., Laskewitz A., Faas M.M., Scherjon S.A., Erwich J.J.H.M., Gordijn S.J., Prins J.R. Journal of Immunology Research 2018; 1–6
Part 2 Pathology: Altered Adaptations of Memory T Cells 117
in Preeclampsia
6. Lower activation of CD4+ memory T cells in preeclampsia 119 compared to healthy pregnancies persists postpartum
Kieffer T.E.C., Scherjon S.A., Faas M.M., Prins J.R. (Submitted)
7. Decidual Memory T Cell Subsets and Memory T Cell 141
Stimulatory Cytokines in Early- and Late-Onset Preeclampsia
8. Review: Immunomodulators to treat recurrent miscarriage 179
Prins J.R., Kieffer T.E.C., Scherjon S.A. European Journal of Obstetrics and Gynecology and Reproductive Biology 2014; 181:334-337.
9. Prednisolone treatment during early pregnancy disrupts maternal 191 immune adaptation and modulates offspring development in mice
Kieffer T.E.C., Chin P.Y., Green E.S., Moldenhauer L.M., Prins J.R., Robertson S.A. (Manuscript in preparation)
10. General Discussion 217
Part 4 Appendices 233
English Summary 234
Dutch Summary (Nederlandse samenvatting) 239
List of Publications 245
List of Contributing Authors 246
Acknowledgements (Dankwoord) 250
General Introduction
1
GENERAL INTRODUCTION
Pregnancy is an exceptional immunological phenomenon. Normally, the immune system generates an immune response upon activation by non-self antigens to clear the non-self antigen presenting body1. However, during pregnancy specific immune adaptations prevent this immune response towards fetal-paternal antigens2,3, while immunity towards pathogens remains intact4. Thus, the maternal immune system adapts to prevent rejection of the semi-allogeneic cells of the fetus and with that fetal-maternal immune tolerance is generated.
Insufficient adaptations of the maternal immune system may contribute to dysfunc-tional fetal-maternal immune tolerance, which is implicated in the pathophysiology of pregnancy disorders such as infertility, pregnancy loss, fetal growth restriction, and preeclampsia5–7, which affect both mother and child. The exact mechanisms responsible for fetal-maternal immune tolerance are incompletely understood. Studies into the immunology of pregnancy aim to elucidate the function and dysfunction of these mechanisms. Ultimately, these studies will contribute to development of medical interventions, possibly through modulation of the immune response, to prevent or treat pregnancy disorders.
The concept of fetal-maternal immune tolerance
After previous studies implying immunologic tolerance by Ray Owen8,9, in 1953, Sir Peter Medawar was the first to conclusively show experimentally induced immunologic tolerance in mice10. In the same year, he acknowledged the unique phenomenon of the allogeneic fetus being carried by the mother without rejection and posed:
“How does the pregnant mother contrive to nourish within itself, for many weeks or months, a foetus that is an antigenically foreign body?”11. Medawar hypothesized that fetal-maternal tolerance is explained by three mechanisms. Firstly, by physical separation between the mother and fetus, secondly by antigenic immaturity of the fetus, and thirdly, by immunological inertness of the mother. However, it has now conclusively been shown that the three hypotheses of Medawar are invalid12–16.
Firstly, absolute physical separation between the mother and fetus does not exist. It is known that fetal cells are in direct contact with the maternal blood and even per-sist in the maternal circulation many years after pregnancy, i.e. microchimerism12,13. Secondly, the maternal immune system responds to fetal cells14, showing that antige-nic immaturity is not a valid explanation for immune tolerance. Thirdly, Medawar’s
statement, regarding maternal inertness, has been shown to be incorrect as the mater-nal immune system is still able to generate an immune response towards pathogens during pregnancy4, and fetal-antigen specific lymphocytes have been shown which are able to generate fetal-antigen specific tolerance15,16. Thus, the maternal immune system actively responds to fetal antigens, does not reject fetal cells, and actively establishes fetal-maternal immune tolerance.
After Medawar, numerous studies attempted to explain the mechanisms of maternal tolerance towards the fetus. Although our knowledge on immunology of pregnancy has improved, the exact mechanisms are still incompletely understood.
Immunology of pregnancy
As described above, fetal trophoblasts are in direct contact with maternal immune cells in decidual tissue of which two subtypes are distinguished17,18. The uterine lining located around the fetal membranes is called the decidua parietalis and interacts with chorionic trophoblasts17. The decidua basalis is part of the placenta and is invaded by extravillous trophoblasts and due to its anatomic location has a different blood supply18. Interaction of the maternal immune cells with fetal trophoblasts results in recruitment of specific immune cells from the blood19. Due to different amounts of antigen exposure, cell recruitment, and blood supply the immune cell repertoire greatly varies between different tissues during pregnancy. Fetal syncytiotrophoblasts cells are in contact with the maternal blood circulation and therefore with circulating maternal immune cells20. This indicates that specific adaptations are also needed in circulating maternal immune cells.
Adaptations of both the innate and adaptive immune system are required for maintaining fetal-maternal immune tolerance and pregnancy success21. The innate immune system is the first line of immune defence of the human body and consists of epithelia, phagocytic cells (monocytes, neutrophils, and macrophages), natural killer (NK) cells, blood proteins (inflammatory mediators and parts of the complement system) and cytokines1. Adaptations of innate immune cells in pregnancy include activation, increased numbers and altered function of monocytes3,22, granulocytes23, and NK cells24. Together with many more adaptations, the innate immune system greatly attributes to fetal-maternal tolerance in the peripheral circulation and at the fetal-maternal interface, where they are especially important in the processes of spiral artery remodeling and placental development25. This thesis focuses on the adaptive
The humoral immune response protects against extracellular micro-organisms and is managed by B-lymphocytes (B cells) mostly1. The main mediators of cellular immunity are T lymphocytes (T cells)1,26. T cells are the most studied immune cells in pregnancy and the main interest of this thesis26.
T cells develop from haematopoietic stem cells in the bone marrow and mature in the thymus in the juvenile, thereafter the T cell population is maintained by division of mature T cells outside the primary lymphoid organs1,27. They are identified by their T cell receptor (TCR) which recognizes antigens presented in the context of major histocompatibility complexes (MHC) by other cells28. Different types of T cells are distinguished according to the class of MHC the cell responds to. CD8+ T cells, or cytotoxic T lymphocytes, recognize antigens on MHC class I molecules expressed on all nucleated cells and platelets, whereas CD4+ T cells, or helper T lymphocytes, respond to antigens presented on MHC class II molecules expressed on antigen presenting cells such as macrophages and dendritic cells28. CD4+ cells can produce cytokines and thereby affect the response of several other immune cells, coordinating the immune response1. Most of the CD8+ cells are considered cytotoxic and act by killing cells through secretion of pro-inflammatory cytokines or through apoptosis induction by cell-cell interaction with the target cell1.
Figure 1. Schematic overview of the anatomy of the placenta, decidual tissue, and membranes. Source: Fleischer’s Sonography in Obstetrics and Gynecology: Textbook and Teaching Cases, 8th edition; Chap-ter 7: Placenta, Cord, and Membranes; Authors: J.M. Mastrobattista, E.C. Toy. Copyright © 2018 by McGraw-Hill Education.
Based on their effector function, the CD4+ cell compartment is divided into several subsets. In the CD8+ cell compartment on the other hand, subsets based on effector function are rarely used29. The CD4+ cell compartment is subdivided into several subsets of which the T helper 1 (Th1), Th2, Th17, and T regulatory (Treg) cell sub-sets, are the mostly studied1. Expression of transcription factor TBET, induces diffe-rentiation of T cells towards Th1 cells which are known for their pro-inflammatory immune response towards bacteria and protozoa, and are characterized by IFNγ secretion30,31. Th2 cells, differentiated from effector T cells with over expression of transcription factor GATA3, manage the immune response towards parasites and produce interleukin-4 (IL4), IL5, and several other cytokines30,31. They can also regulate anti-inflammatory processes by secretion of IL10 which can suppress the Th1 response and dendritic cell function32. Th17 cells are recognized by RORγT expression and IL17 production and therewith generate a pro-inflammatory response and inhibit Treg cell differentiation33–35. Treg cells have immune regulatory and anti-inflammatory abilities and are of particular interest in fetal-maternal tolerance36,37. Treg cells are studied in more detail in this thesis and are more elaborately introduced below38.
T cells are also divided into naïve cells, effector cells, and memory cells39. This subdivision is made in both CD4+ and CD8+ T cell subsets and is based on antigen experience of the cells39. The naïve T cell population represents the cells that have never been activated through antigen exposure. Effector cells have been activated by antigens and are executing effector functions, whereas memory T cells were pre-viously activated by antigen and remain waiting for a secondary encounter with the same antigen to elicit a more enhanced response39. Increasing evidence implies a role for memory T cells in fetal-maternal tolerance and these cells are therefore further studied in this thesis40. Immune cell subsets are also characterized by specific migra-tory patterns41. Whereas some immune cells only circulate in peripheral blood and lymph nodes, other immune cells stay resident in one tissue their entire lifespan42,43.
T regulatory cells
Treg cells in the CD4+ cell lineage are identified by transcription factor forkhead box p3 (FOXP3), and are also known to express high levels of CD25 (IL2Rα) and low levels of CD127 (IL7Rα)44. Most studies focus on CD4+ Treg cells, but Treg cells with a CD8+ background are also described45,46. CD8+ Treg cells are not identified by Foxp3 expression but mainly by their immune regulatory cytokine secretion profile45,47. They are less prevalent, more difficult to identify, and are not studied much in
CD4+ Treg cells are capable of suppressing CD4+, CD8+ and B cell proliferation and cytokine secretion through several pathways such as secretion of IL10 and transforming growth factor beta (TGFβ) and expression of CD3948–52. Together with their inhibiting effects on antigen presenting cells, such as dendritic cells and macrophages, they have controlling abilities on both the adaptive and the innate immune response53,54. Their immune regulatory function is implicated in physiological and pathological contexts such as gastro-intestinal homeostasis55, auto-immune diseases56, respiratory disorders57, and oncological pathology58.
Aluvihare et al. was the first to describe the essential role for Treg cells in fetal-ma-ternal tolerance by showing rejection of semi-allogeneic fetuses in Treg cell depleted mice36. Later, it was reported that adaptations of the Treg cell population occur even before embryo implantation and are essential for reproductive success59. Robertson et al. showed that seminal fluid is one of the mediators of the accumulation of Treg cells in the mouse uterus before implantation60,61. Recently, Treg cell abundance in the mouse uterus in early pregnancy was found to be involved in uterine artery function and decreasing oxidative stress62.
The importance of Treg cells in successful reproduction was also found in humans37,63. In healthy human pregnancy, Treg cell numbers increase, with a peak in the second trimester, and a clear decline in the weeks before spontaneous labor64–66. Insufficient adaptations of the Treg cell population in human pregnancy are implicated in the pathophysiology of many complications of reproduction as infertility38,67, pregnancy loss68, and preeclampsia38,69,70. In preeclampsia, decreased numbers and Treg cells with dysfunctional immune regulatory function are associated with a more pro-inflam-matory state towards the fetal-placental unit70–72.
Upon antigen exposure, the Treg cell population expands and after the immune response most Treg cells perish through apoptosis46. However, a population of Treg cells differentiates into Treg memory cells and persists in either secondary lymphoid organs, the blood circulation or remain resident in the tissue73–75. Treg memory cells are further discussed in chapter 2 of this thesis.
Memory T cells
Immunologic memory is defined as the ability of the immune system to remember anti-gens and mount a response of greater magnitude and faster kinetics on a secondary
encounter with the same antigen76. Immunologic memory is formed by T cells, B cells and recently natural killer cells were also found to have memory capabilities77–79.
Memory T cells are formed during an immune response. Whereas most effector T cells die, some effector T cells differentiate into memory T cells and remain present to rapidly re-accumulate upon a secondary encounter with the memorized antigen80. The secondary response elicits a much quicker expansion of the T cell population, higher levels of cytokine secretion, and faster elimination of the allogeneic body80.
In pregnancy, long time surviving memory T cells are formed with specificity for fetal-paternal antigens81–83. However, instead of quick elimination of fetal cells upon a second encounter with the fetal-paternal antigens, in pregnancy, immune tolerance towards the fetal cells is required for pregnancy success. Increasing evidence shows that altered numbers and function of memory T cells in pregnancy are important for pregnancy success and a beneficial role is suggested16,81,84,85. Moreover, insufficient adaptations of memory T cell populations are implicated in the pathophysiology of pregnancy complications such as preeclampsia70,81,84,86. In chapter 2 memory T cells and their subsets are introduced and their involvement in pregnancy and in pregnancy complications are discussed.
Preeclampsia
Preeclampsia is characterized by de-novo hypertension, together with either pro-teinuria, maternal organ dysfunction (including liver involvement, neurological com-plications, or haematological complications), or fetal growth restriction87, though symptomless pre-clinical stages precede88. This hypertensive disorder complicates 2-8% of pregnancies89. Together with gestational- and chronic hypertension, it affects 5-10% of all pregnancies and causes 3-5% of maternal deaths in high-income coun-tries and up to 26% in Latin America and the Caribbean90–92. Its associated fetal growth restriction and (iatrogenic) preterm birth are associated with an increase in fetal morbidity and mortality93. Besides an increased risk of preeclampsia in a subse-quent pregnancy, long term effects of preeclampsia for women with a preeclamptic pregnancy in their obstetric history include hypertension, cardiovascular diseases, diabetes mellitus, and renal dysfunction93–95. Long term effects on offspring are not studied as much as long term maternal effects, but reduced cognitive performance96,97, and increased risks of high blood pressure and stroke in adolescence have been shown93,98.
After decades of research, the multifactorial pathophysiology of preeclampsia is still incompletely understood. Evidence of several pathways is reported, and most likely each case has a unique combination of factors in its aetiology with involvement of multiple mechanisms that work intertwined. As an attempt to categorize different types of preeclampsia by its aetiology, early-onset and late-onset preeclampsia are distinguished99. Early-onset, defined as diagnosis before 34 weeks of gestation, is considered to be mainly caused by disturbance in placentation and is associated with fetal growth restriction100,101. Late-onset preeclampsia, diagnosed after 34 weeks of gestation, is supposedly associated with ageing of the placenta and reduced intervillous perfusion and does not cause fetal growth restriction in most cases100,101.
It is proposed that the preclinical stage of early-onset preeclampsia starts early in pregnancy with abnormal invasion of trophoblast cells in the myometrium102. Poor placentation, as demonstrated by insufficient spiral artery remodelling, may lead to insufficient uteroplacental circulation103. The clinical stages of preeclampsia are most likely caused by oxidative stress due to factors coming from the dysfunctional placenta resulting in a systemic inflammatory response of the mother104. It is proposed that one of the underlying dysfunctions in preeclampsia would be insufficiency of the maternal immunologic adaptations to tolerate the fetal cells105. Presumably, the immune cells do not adequately regulate the processes in early pregnancy such as myometrial trophoblast invasion, essential for normal implantation and remodelling of the spiral arteries to form a low resistance flow network supplying the intervillous space105. Instead, they induce an inflammatory response towards the fetal placental unit, possibly resulting in pregnancy induced hypertension in mild cases, to pree-clampsia and pregnancy loss when more severe100,104.
A great variety of immune cells from both the innate and adaptive immune system are implicated in the pathophysiology of preeclampsia. As indicated above, the immune response during normal pregnancy has to adapt in order to tolerate the semi-allogeneic fetus. However, during preeclampsia this adaptation may fail21,105, resulting in aberrant immune responses during preeclampsia. For instance, inadequate maternal NK cell interaction with fetal antigens in early pregnancy has been found to induce insufficient immune regulation of placental development106. Reduced Treg cell numbers, as found in preeclampsia69,71, may cause the imbalance in Th1, Th2, and Th17 cells which results in secretion of pro-inflammatory cytokines and aberrant activation of innate immune cells3, resulting in the systemic inflammatory response as observed in preeclamptic women105.
Epidemiologic evidence implies a role for immunologic memory in the pathophysio-logy of preeclampsia. Where at first it was believed that preeclampsia was a disease of first-time mothers, in 1994 it was found that the lower risk of preeclampsia in a subsequent pregnancy was lost with a change of partner and was thus associated with primipaternity107,108. Thereafter, it was shown that a longer period of regular exposure to seminal fluid to the vaginal or oral mucosa before conception contributes to a lower risk of early-onset preeclampsia109–111. These findings suggest that immuno-logic memory, and possibly memory T cells, have beneficial effects during pregnancy.
Aim and outline of this thesis
This thesis aims to analyse the adaptations of memory- and Treg cells in healthy- and complicated pregnancies, and to explore the effects of modulation of the immune response on pregnancy and pregnancy outcome. We aim to increase knowledge on memory- and Treg cells in healthy pregnancies and preeclampsia.
In this thesis, memory T cells and / or regulatory T cells are investigated in preg-nancy in three different settings; physiology, pathology, and after immune modulatory treatment such as prednisolone.
In chapter 2 a detailed introduction on the current state of the art knowledge on memory T cells in pregnancy is provided.
Part 1 starts with chapter 3, which gives insight into the persistent effects of normal
human pregnancy on the memory T cell (sub)populations in peripheral blood of healthy women. In chapter 4 focus will be on memory T cell populations at the fetal-mater-nal interface in healthy pregnancies, and alterations of memory T cells in decidual tissue of first pregnancies compared to pregnancies of women who delivered before. In chapter 5, alterations of the maternal immune response in early pregnancy asso-ciated with fetal sex are described. This chapter emphasizes the importance of the consideration of fetal sex in further investigations into the immunology of pregnancy and the delicacy of the immune balance in uncomplicated pregnancies.
In part 2, memory T cells are studied in a pathological condition; preeclampsia. In chapter 6, memory T cell populations are studied in peripheral blood of preeclamp-tic and healthy pregnant women, and women postpartum after a preeclamppreeclamp-tic or healthy pregnancy. In this study, we aimed to show the short- and long term effects of
insight into proportions of memory T cell subsets in preeclampsia, by studying decidual tissue from preeclamptic and uncomplicated pregnancies.
Part 3 of this thesis focuses on modulation of the immune response in pregnancy as
a treatment for pregnancy complications. First, in chapter 8, different possibilities of immunomodulatory treatments to treat recurrent miscarriage are reviewed. In
chap-ter 9, one of the immunomodulatory treatments is investigated further in a mouse
model. The effects of prednisolone treatment in early pregnancy on maternal Treg cells are reported. In addition, the long-term effects on offspring are investigated to gain knowledge on the importance of sufficient maternal immune adaptations in pregnancy for the offspring.
In chapter 10, the findings reported in this thesis are discussed in relation to one another and proposals for future research into memory- and regulatory T cells in pregnancy are proposed.
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2
Memory T Cells in Pregnancy
Frontiers in Immunology 2019; 10:624
Tom E.C. Kieffer
1Anne Laskewitz
2Sicco A. Scherjon
1Marijke M. Faas
2Jelmer R. Prins
11Department of Obstetrics and Gynaecology, University Medical Center Groningen,
University of Groningen, Groningen, the Netherlands
2Division of Medical Biology, Department of Pathology and Medical Biology,
ABSTRACT
Adaptations of the maternal immune response are necessary for pregnancy success. Insufficient immune adaption is associated with pregnancy pathologies such as infer-tility, recurrent miscarriage, fetal growth restriction, spontaneous preterm birth, and preeclampsia. The maternal immune system is continuously exposed to paternal-fetal antigens; through semen exposure from before pregnancy, through fetal cell exposure in pregnancy, and through microchimerism after pregnancy. This results in the genera-tion of paternal-fetal antigen specific memory T cells. Memory T cells have the ability to remember previously encountered antigens to elicit a quicker, more substantial and focused immune response upon antigen reencounter. Such fetal antigen specific memory T cells could be unfavorable in pregnancy as they could potentially drive fetal rejection. However, knowledge on memory T cells in pregnancy has shown that these cells might play a favorable role in fetal-maternal tolerance rather than rejection of the fetus. In recent years, various aspects of immunologic memory in pregnancy have been elucidated and the relevance and working mechanisms of paternal-fetal antigen specific memory T cells in pregnancy have been evaluated. The data indicate that a delicate balance of memory T cells seems necessary for reproductive success and that immunologic memory in reproduction might not be harmful for pregnancy. This review provides an overview of the different memory T cell subtypes and their function in the physiology and in complications of pregnancy. Current findings in the field and possible therapeutic targets are discussed. The findings of our review raise new research questions for further studies regarding the role of memory T cells in immune-associated pregnancy complications. These studies are needed for the identification of possible targets related to memory mechanisms for studies on pre-ventive therapies.
INTRODUCTION
Immune tolerance towards paternal-fetal antigen is crucial for reproductive success since dysfunctional tolerance is implicated in the pathophysiology of pregnancy com-plications as infertility, recurrent miscarriage, fetal growth restriction, spontaneous preterm birth, and preeclampsia1–4. In reproduction, the maternal immune system is exposed to paternal-fetal antigens (Figure 1). Firstly, the male antigen is introduced to the maternal immune system through semen exposure even before pregnancy5. Secondly, paternal-fetal antigens are exposed at the fetal-maternal interface in preg-nancy since the maternal immune cells in blood are in direct contact with fetal trophoblast cells in the placenta6,7. Additionally, in pregnancy, there is trafficking of
fetal cells expressing paternal-fetal antigens to maternal tissues at low levels which can recirculate in the maternal blood for years after pregnancy8,9. This phenomenon is called microchimerism8,9. It has been shown that the exposure of the maternal immune system to paternal-fetal antigens induces a memory T cell population with paternal-fetal antigen specificity10–12.
The memory lymphocyte population is comprised of memory T lymphocytes (T cells) and memory B lymphocytes (B cells)13,14. Memory T cells are the most studied and appear to be the most important memory cell population in reproduction. Memory cells enable the immune system to protect the body from pathogens efficiently by generating a more adequate immune response to a known antigen, making it unne-cessary to elicit a new response to an antigen that was encountered before15. This
Figure 1. Hypothesis on generation of the memory T cell population in reproduction through paternal-fetal antigen exposure. Firstly, naive T cells are exposed to the male antigen through antigens in seminal fluid. A subsequent encounter with the antigens occurs during pregnancy through exposure to fetal antigens on trophoblast cells and through microchimerism. Postpartum, the maternal immune system remains exposed to fetal antigens through microchimerism. In addition, postpartum, memory T cells are possibly exposed to paternal antigens through exposure to seminal fluid. In a subsequent pregnancy, the maternal memory T cells likely reaccumulate and respond to the cognate paternal-fetal antigens.
diseases and more recently to fight cancer and auto-immune diseases16–18. In gene-ral, a more aggressive immune response towards pathogens is protective for health since the pathogen is cleared faster, however, the same aggressive response towards paternal- or fetal antigens would be disastrous for fetal and maternal health. Indeed, most studies of memory T cell populations in reproduction indicated that memory T cell subsets may exhibit a different function, proliferation pattern and migratory abilities towards paternal antigens in healthy pregnancies as compared with their function, proliferation and migratory abilities towards other antigens12,19,20. In fact, specific memory cell populations have been shown to be involved in generating immune tolerance, rather than immune rejection, towards paternal-fetal antigens12,21–23.
In recent years, the implication and relevance of memory T cells in pregnancy and complications of pregnancy have been revealed. Major conceptual breakthroughs were seen in the T cell field, showing the role of memory T cells in reproductive fit-ness in mouse studies11,12,21. Since increasing numbers of human studies on memory T cells have been published, this review gives an overview of the current literature on the different memory T cell subtypes and their adaptation in pregnancy and the implication of memory T cells in different complications of pregnancy. We will mainly focus on human studies and refer to mouse studies if needed. Current research gaps, controversies and possible therapeutic targets will also be discussed.
MEMORY T CELLS
The memory T cell population is formed during a primary antigen response24. In the primary response, antigens are presented to T cells through major histocompatibility complex (MHC) molecules25. Depending on the type of MHC molecule, either type I or type II, CD8 positive or CD4 positive T cells respectively are activated through the T cell receptor (TCR) on the cell membrane25. Additional co-stimulatory molecules can connect to co-stimulatory receptors on the T cell such as CD28 and CD70, for extra induction of the T cell response25,26. Depending on the cytokine environment, CD4+ cells differentiate into either different T helper (Th) subsets (Th1, Th2, and Th17) which help in inducing/activating immune responses through secretion of cytokines, or into T regulatory (Treg) cells which exert regulatory effects on other immune cells after activation27. After the primary response, most CD4+ cells die, but some CD4+ cells differentiate into CD4+ memory T cells24,28. CD8+ cells also differentiate into different subpopulations; i.e. effector CD8+ cells which are ready to release cytotoxic cytokines or induce apoptosis via cell surface interaction, and a small population of regulatory
CD8+ cells which exhibit an immune regulatory function29. Once the pathogen is cleared, most CD8+ cells die, however some proliferate into memory CD8+ cells29.
Several memory T cell subsets are known, and can be distinguished by various markers (Table 1 and Table 2). The main markers are CD45RO expression, and lack of CD45RA expression30,31. The CD45RO+CD45RA- phenotype has been linked to long living memory T cells30,31. It should be noted that CD45RO expression and lack of CD45RA expression are not conclusive markers for memory T cells, since their expression does not predict long time survival and rapid effector function upon secondary exposure per se32. In addition, it has been shown that CD45RO+ T cells can be reprogrammed and go back to a CD45RO- naive phenotype33,34. So far there are no other reliable markers of phenotype memory T cells in clinical experiments, therefore, phenotypic characterization of the memory cell population by CD45RO expression is widely used. Memory CD4+ and CD8+ cells can be divided into subsets based on their migration pattern, cytokine secretion abilities, and protein expression profile. The main memory cell subsets are the central memory (CM) cells and the effector memory (EM) cells, although the number of subsets is expanding rapidly (Table 1 and Table 2). The CM cell subset differentiates into effector cells upon secondary antigen exposure and is characterized by CCR7 expression which makes them home to secondary lymphoid organs31,35. The EM cell subset is characterized by their presence in peripheral tissue and direct pro-inflammatory effector function upon secondary antigen encounter with the cognate antigen31. Below, an overview of the current knowledge of the various memory T cell subsets in pregnancy is reviewed.
CD4
+MEMORY CELLS IN PREGNANCY
Within the CD4+ memory cell population, a subdivision has been made based on migration pattern and effector function; i.e. CD4+ effector memory (CD4+ EM) cells, CD4+ central memory (CD4+ CM) cells, CD4+ tissue resident memory (CD4+ TRM) cells, CD4+ T follicular helper memory (CD4+ FHM) cells, CD4+ regulatory memory cells, and CD4+ memory stem cells36–43.
It has been known for many years that pregnancy and some pregnancy complica-tions affect the general CD4+ memory T cell population. In 1996, it was shown that general CD4+ memory cell (CD4+CD45RO+) proportions in peripheral blood were lower from the second trimester onwards until 2-7 days postpartum compared to pro-portions in non-pregnant controls44. These findings have been followed up by studies
proportions of total memory T cells in peripheral blood have been found compared to healthy pregnant controls. Early studies also showed CD4+CD45RO+ memory cells in the decidua and showed that CD45RO expression on CD4+ cells is upregulated in the decidua compared to CD4+ cells in peripheral blood50,51. Later, Gomez-Lopez et al. suggested a role for CD4+ memory cells in human term parturition by showing an increase of CD4+ memory cells (CD4+CD45RO+) using immunohistochemistry on choriodecidual tissue from women in spontaneous labor at term compared to women with term scheduled caesarean sections52. The early data already indicated that memory T cells are affected by pregnancy and its complications. In more recent years, studies have focused on specific subsets of CD4+ memory cells. These data are reviewed per memory T cell subset below.
CD4+ effector memory cells in pregnancy
Th1, Th2, and possibly Th17 effector cells can differentiate into CD4+ EM cells53–55. CD4+ EM cell characterization is based on the lack of expression of lymph node homing receptors CC-chemokine receptor-7 (CCR7) and CD62L (L-selectin), which enables them to migrate to peripheral tissue37. EM cells are the memory cells with the fastest immune response on a secondary encounter. Within several hours after re-stimulation with a memorized antigen, CD4+ EM cells produce a variety of cytoki-nes as interferon-gamma (IFN-gamma), tumor necrosis factor (TNF), interleukin-4 (IL4) and IL531,55,56. A specific subtype of CD4+ EM cell can re-express CD45RA after anti-gen stimulation (TEMRA)57. These cells are poorly studied and there are no published investigations on CD4+ TEMRA cells in reproduction to our knowledge.
In the second and third trimesters of pregnancy, two studies showed higher CD4+ EM cell (CD45RA-CCR7- and CD45RO+CCR7-) proportions in peripheral blood, com-pared to proportions of these cells in non-pregnant women23,58, while another study found decreased numbers of CD4+ EM cells in peripheral blood during pregnancy59. Differences between the studies could be due to the fact that that hormonal fluctuations during the menstrual cycle were not taken into account in the latter study. Not only is the proportion of CD4+ EM cells increased during pregnancy, these cells also showed increased expression of CD6958, as well as decreased expression of programmed death-1 (PD-1)23. This suggests that there is increased activation of CD4+ EM cells, and that these cells are less susceptible to apoptosis. The increase of CD4+ EM cells is not only seen during pregnancy, but also years after when CD4+ EM cell proportions remained increased, i.e. at gestation levels as compared with women that have never been pregnant58. These cells also showed increased CD69 expression after pregnancy,
which could suggest persistent activation through exposure to antigen. This could be related to microchimerism, although it remains to be investigated whether the increased EM cell proportion is due to an increase in cells specific for paternal-fetal antigens.
Whereas in blood the proportion of CD4+ EM cells of the total CD4+ cell population was about 20-30%19,58,60, locally, in the decidua, the proportion of CD4+ EM cells (CD45RA-CCR7-) was higher with 50-60% of the total CD4+ cell population being EM cells19,60. This may indicate accumulation of CD4+ EM cells in the decidua, alt-hough it can also be simply due to the fact that naive T cells do not accumulate in peripheral tissue61. Important for the function of memory T cells is the expression of co-stimulatory molecules like CD2862. Such molecules are important for the recall response of memory T cells63. Interestingly, within the CD4+ EM cell population in the decidua, the proportion of the EM subset not expressing co-stimulatory molecules is highly increased compared to peripheral blood19, suggesting that the CD4+ EM cells in the decidua may not be able to mount a secondary response comparable to CD4+ EM cells in peripheral blood. Despite this, increased IFN-gamma and IL4 expressions were found in decidual CD4+ EM cells compared to CD4+ EM cells in peripheral blood in vitro following mitogen stimulation19. This may be related to the high local progesterone concentrations at the fetal maternal interface19. The decidual EM cells were not only able to respond to mitogen stimulation, they were also able to respond to fetal antigens19. The fact that the decidual EM cells are able to respond to fetal antigens and other stimuli suggests that there are extrinsic or intrinsic mechanisms at the fetal-maternal interface to suppress these cells. One of these mechanisms could be the presence of Treg cells64,65. Another mechanism may be the expression of immune inhibitory checkpoint receptors on decidual CD4+ EM cells19. Activation of these receptors inhibit immune responses to avoid autoimmunity and chronic inflam-mation66. Increased expression of the immune inhibitory checkpoint receptors PD-1, T cell immunoglobulin and mucin domain 3 (Tim-3), cytotoxic T lymphocyte antigen 4 (CTLA-4) and lymphocyte activation gene 3 (LAG-3), on CD4+ EM cells in the decidua was found as compared to peripheral blood19. These findings are in line with Wang et al. who showed that the majority of CD4+ EM cells (CD44+CD62L-) in first trimester decidual tissue from healthy terminated human pregnancies, expressed Tim-3 and PD-167. A role for such immune inhibitory check point receptors in pregnancy has been shown in mouse studies67. Blocking the Tim-3 and PD-1 pathway (not on CD4+ EM cells specifically) in healthy pregnant mice showed that lower expression of Tim-3 and PD-1 increased fetal resorption rates67. These studies propose a regulatory function for CD4+ EM cells locally that could be favorable for fetal-maternal immune
The current data on CD4+ EM cells in women with uncomplicated pregnancy out-comes show that during pregnancy CD4+ EM cells may accumulate in the decidua and remain present at higher levels and higher activated proportions in peripheral blood postpartum58. In addition, the CD4+ EM cell population in the decidua has a different phenotype with increased IFN-gamma expression, however the CD4+ EM cell population also has increased expression of immune inhibitory proteins compared to peripheral blood19. To understand the relevance and function of CD4+ EM cells in fetal-maternal tolerance and their role in the postpartum period, further research should focus on their general and more specifically on their antigen specific function, since none of the studies has shown antigen specific tolerance induction by CD4+ EM cells yet.
Unfortunately, until now, CD4+ EM cells have been hardly studied in complications of pregnancy. CD4+ EM cells were studied in preeclampsia by Loewendorf et al. who performed flow cytometric analyses on peripheral blood and a swab from the intrauterine cavity during caesarean sections68. They did not find differences in levels of CD4+ EM cells between healthy and preeclamptic women in peripheral blood or in lymphocytes isolated from the intra uterine swab68. However, since the specific tissue of origin of the cells from the swab cannot be defined, caution should be taken when interpreting these results. In non-pregnant women suffering recurrent spontane-ous miscarriages, higher proportions of EM cells were observed in peripheral blood compared to non-pregnant fertile controls69. This study did not further specify the CD4+ or CD8+ status or phenotype. With the proposed relevance of CD4+ EM cells in fetal-maternal tolerance it would be of great value to gain knowledge on CD4+ EM cells in complications of pregnancy.
CD4+ central memory cells in pregnancy
CD4+ CM cells circulate in the blood and are home to lymph nodes through expres-sion of lymph node homing receptors CCR7 and CD62L35–37. CD4+ CM cells secrete IL2 and only very low levels of effector cell cytokines28,31. Upon secondary antigen exposure, or spontaneously, in the presence or absence of polarizing cytokines, CD4+ CM cells differentiate into Th1, Th2 and CD4+ EM cells, and produce effector cytokines as IFN-gamma and IL431,70–72. Furthermore, CM cells can quickly cause expansion of the antigen specific T cell population72.
During pregnancy, as for CD4+ EM cells, CD4+ CM cells are studied mainly in the circulating blood and less at the fetal-maternal interface. One study looked at CD4+ CM cells (CD45RA-CCR7+) in decidual tissue at the end of pregnancy and showed
that proportions of CD4+ CM cells were higher compared to peripheral blood from non-pregnant women60. Another study evaluated first trimester decidual tissue from terminated healthy pregnancies, and showed that about 40% of CD4+ CM cells (CD44+CD62L+) were Tim-3+ and PD-1+67. This appears to be a subset of CD4+ EM cells that have a strong suppressive capacity on proliferation and preferentially pro-duce Th2 type cytokines67. Since blocking of PD-1 and Tim-3 in pregnancies in mice induced fetal loss67, the Tim-3+PD-1+ CD4+ EM cells may be important for maintaining normal pregnancy.
A number of studies in pregnancy observed that the proportions of CD4+ CM cells (CD45RA-CCR7+) in peripheral blood are comparable between women in the second or third trimester of pregnancy and in healthy non-pregnant women23,58,59. However, it seems that after pregnancy the CD4+ CM cell proportions in peripheral blood are increased, since CD4+ CM cells were higher in women after pregnancy compared to pregnant women and compared to women that have never been pregnant58. Whether the CD4+ CM cells are activated in the circulation of pregnant women remains to be established, since expression of the activation marker CD69 was higher during pregnancy as compared with non-pregnant women58, whereas expression of the activation markers HLA-DR and CD38 was not affected in the CD4+ CM cell population (CCR7+CD45RO+) in peripheral blood from 3rd trimester pregnant women compared to non-pregnant women59. This higher CD69+ proportion of CD4+ CM cells in pregnancy remained high in women after pregnancy compared to women who have never been pregnant58.
To date, CD4+ CM cells are investigated in two complications of pregnancy, i.e. preeclampsia and miscarriages. In preeclampsia, slightly, but significantly higher proportions of CD4+ CM cells (CD45RO+CCR7+) were found in peripheral blood from preeclamptic women compared to healthy pregnant women68. Proportions of CD4+ CM cells isolated from a swab from the intrauterine cavity during a caesarean section did not show differences between preeclamptic and healthy pregnant women68. This study also analyzed expression of co-stimulatory molecules, CD28, CD27 and the survival receptor CD127 (IL7Ralpha chain), on CD4+ CM cells68. Only a difference in CD28 expression was found: in an intrauterine swab from preeclamptic women, CD4+ CM cells expressed lower levels of CD28 compared to healthy pregnant women68. In peripheral blood this difference was not observed68. In women suffering from recurrent spontaneous miscarriages, higher levels of CM cells (CD45RO+CD62L+) have been found in peripheral blood compared to fertile women69. It was not
