Adaptation and Modulation of Memory and Regulatory T Cells in Pregnancy
Kieffer, Tom Eduard Christiaan
DOI:
10.33612/diss.97355536
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Publication date: 2019
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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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10
GENERAL DISCUSSION
Adaptations of the maternal immune system during pregnancy are crucial for fetal-maternal immune tolerance and pregnancy success since insufficient adaptations are implicated in immune associated complications of pregnancy such as preeclamp-sia1. In the first two parts of this thesis, results are reported which provide insight
into the adaptations of maternal memory- and regulatory T (Treg) cell populations in healthy pregnancy and alterations of these populations in preeclampsia. Understan-ding the immune mechanisms facilitating fetal-maternal tolerance in pregnancy, and its disturbance in preeclampsia might ultimately help to design therapeutic options to treat or prevent immune associated complications of pregnancy. In the last part of this thesis, several immune modulatory treatment options are discussed of which the effects of prednisolone treatment in early pregnancy on maternal immune adap-tations, pregnancy outcome, and offspring development were further investigated in a mouse model.
The present thesis focusses on the fluctuations of memory T cell subsets in healthy pregnancies and preeclampsia. With their ability to elicit a stronger immune response on a second encounter with the same antigen, it seems that these cells might be harmful for fetal health, since in pregnancy there is continuous exposure to fetal-pa-ternal antigens which could result in a more aggressive response and thereby cause pregnancy complications. However, in this thesis we have shown that the number and activation state of memory T cells increase in pregnancy, which appears to be in line with recent data suggesting a beneficial effect of memory T cells in pregnancy and their possible contribution to pregnancy success2–5. Whereas many studies
focussed on the mechanisms of memory T cell homeostasis in the pregnant body5–8,
this thesis focused on differences in memory T cell homeostasis in different groups, i.e. pregnant women, preeclamptic women, and non-pregnant women (before and after pregnancy). By using this approach, insufficient adaptations of memory T cell populations in complicated pregnancies were revealed and persistent changes of the memory T cell populations were observed in women postpartum. This thesis therefore supports the hypothesis that memory T cells play a role in successful fetal-maternal tolerance and that memory T cells might contribute to recurrence risks of pregnancy complications. In figure 1, a hypothesis on the fluctuations of memory T cells before, during and after healthy or complicated pregnancies is shown, based on literature and the results presented in this thesis.
In chapter 3, we reported that pregnancy has persistent effects on memory T cell populations in peripheral blood. In line with other studies, we found higher CD4+
effector-memory (EM) cells in pregnant women compared to nulligravid women9,10.
Figure 1. Hypothetical fl uctuations and CD69 expression of memory T cell populations in peripheral blood and decidual tissue of women with healthy and complicated pregnancies. Nulligravid women
(A) have memory T cells in their peripheral blood. In a healthy fi rst pregnancy (B), the memory T cell population in the peripheral blood and in decidual tissue increases and express CD69. Postpartum after a healthy pregnancy (C), some fetal cells remain latent in the mother’s body (microchimerism), and the memory T cell proportion in peripheral blood remains higher with higher CD69+ proportions compared
to nulligravid women (A). In a subsequent pregnancy (D), higher proportions of the memory T cell popu-lations express CD69+ compared to the fi rst pregnancy (B). When the fi rst pregnancy is complicated by
preeclampsia (E), the memory T cell population in the peripheral blood does reach similar levels to those in healthy pregnant women (B), but they have lower CD69+ proportions compared to healthy pregnant
women. In the decidua of women with complicated pregnancies (E), lower proportions of some memory T cell subsets are observed, accompanied by lower CD69+ proportions compared to healthy pregnant
women (B). Postpartum after a complicated pregnancy (F), some fetal cells remain latent in the mother’s body (microchimerism), and the proportions of memory T cells are comparable with women after a healthy pregnancy (C), but the memory T cell population has lower CD69+ proportions compared to
post-partum women after a healthy pregnancy. In a subsequent pregnancy after a complicated pregnancy (G), the memory T cell populations do not express higher levels of CD69, which might contribute to recurrence of pregnancy complications.
These data could suggest that during pregnancy CD4+ CM and / or CD4+ effector
cells differentiate into CD4+ EM cells, possibly as a result of the presence of the
semi-allogeneic fetus. This may not be surprising, since CD4+ effector and CD4+ CM cells
differentiate into CD4+ EM cells in a regular immune response against a pathogen as
well11. Interestingly, we also observed higher CD4+ central-memory (CM) and CD4+
EM cell proportions in women in the postpartum period compared to nulligravid women. The higher CD4+ EM and CD4+ CM proportions in women years after
deli-very compared to nulligravid women, might be due to an increase of CD4+ CM cells
in late pregnancy or in the postpartum period. Similar to our data which suggest that pregnancy induces memory T cell formation, formation of immunologic memory during pregnancy has been shown by others2,12,13. However, chapter 3, suggests that
this immunologic memory, which is formed during pregnancy, persists postpartum. Next to the persistent changes of memory T cell proportions, in chapter 3, the CD4+ EM and CD4+ CM cell population had higher activated proportions in women
during pregnancy and women postpartum compared to nulligravid women. This is in line with previous studies which showed that normal human pregnancy and also the postpartum period are associated with increased activation of the immune system, which has been found essential for adequate fetal-maternal immune tolerance10,14,15.
In this thesis, we measured T cell activation by identifying CD69 expression on the cell surface. CD69 is expressed on T cells within hours after receiving stimulatory signals through the T cell receptor or through stimulation by cytokines16,17. The duration of
its expression is uncertain and varies in literature between 48 hours up to 72 hours after the stimulus is lost in in vitro studies, but persistent expression has been found in
in vivo studies in chronic disease18,19. The higher CD69 expression on T cells during
pregnancy compared to nulligravid women could be due to exposure to fetal-pater-nal antigens via antigen presenting cells during pregnancy20,21, or due to cytokines
produced by the placenta22.
The higher CD69 expression we found on T cells in women in the postpartum period compared to nulligravid women is more difficult to explain, and a different mechanism can be proposed. Firstly, the higher CD69 expression could be due to an altered cyto-kine milieu postpartum, as has been reported by several others23,24. In chapter 6, we
investigated the cytokine milieu in women postpartum as well, however, in this study our goal was to assess the differences between formerly preeclamptic and formerly healthy pregnant women, and we did not measure the cytokine levels in nulligravid women. It would be of interest in future studies to analyse whether the cytokine milieu postpartum contributes to the higher activated proportions of memory T cells
in women postpartum compared to nulligravid women as described in chapter 3. Secondly, the higher activated memory T cell proportions postpartum could also be caused by repeated activation of the memory T cell population postpartum through microchimerism, which is the circulation of fetal cells in maternal tissues and blood after pregnancy25. This could be important, since continuous low activation of memory
T cells by the memorized antigen has been found to be beneficial for maintaining a sufficient memory T cell population26. However, continuous stimulation of the memory
T cell population through microchimerism seemed less likely after finding lower CD69+
memory T cell proportions in peripheral blood of formerly preeclamptic women com-pared to formerly healthy pregnant women in chapter 6. Preeclampsia is associated with higher concentrations of fetal microchimerism in the mother27, and if increased
memory T cell activation would be caused by microchimerism, preeclampsia would likely be associated with higher CD69+ proportions postpartum, which is the
oppo-site of what we found. In short, the underlying mechanism for the persistently higher CD69+ memory T cell proportions after healthy pregnancies remains unknown, and
would be interesting to focus on in future studies.
After showing persistent changes of the memory T cell population and its CD69 expres-sion in women postpartum after a healthy pregnancy in chapter 3, we speculated on the formation of immunologic memory during pregnancy and a possible implication for this in a subsequent pregnancy. One implication could be that memory T cells might play a role in lowering the risk of pregnancy complications in a subsequent pregnancy, and therewith explain the epidemiologic findings of pregnancy complica-tions occurring in first pregnancies more frequently than in subsequent pregnancies, especially when from the same partner28,29. Previous studies by others showed more
extensive trophoblast invasion30, and better spiral artery development in multigravid
women compared to primigravid women31, which could possibly be due to a more
favourable immune environment and its associated processes in multigravid women. We speculate that immunologic memory formed in a first pregnancy might support immune adaptation in a subsequent pregnancy, thereby for instance improving spiral artery development and trophoblast invasion, which are processes guided by immune cells. Therefore, in chapter 4, we investigated memory T cell populations present in the decidua from primigravid women and from women who had been pregnant before. Our main finding was higher expression of CD69 on every T cell subset analysed in this study in the decidua parietalis of healthy multigravid women compared to healthy primigravid women. In combination with the findings of lower CD69+ proportions of
several memory T cell subsets in the decidua of preeclamptic women compared to healthy pregnant women in chapter 7 and the findings of lower CD69+ proportions
on memory T cells in formerly preeclamptic women compared to formerly healthy pregnancy women in chapter 6, questions are raised whether CD69 expression is beneficial for pregnancy success.
It was proposed that the cause for the higher CD69+ proportions of memory T cells
in multigravid women compared to primigravid women in chapter 4 is reactivation of memory cells by the cognate fetal-paternal antigen in multigravid women. This reactivation might elicit an immune response with higher CD69+ cell proportions in
multigravid women compared to primigravid women who presumably do not have a similar immunologic memory. This suggestion is in line with a report showing that CD69 expression on memory T cells upon reactivation is more substantial compared to CD69 expression on memory T cells during the initial response32. Comparison of
CD69+ proportions between multigravid women pregnant from a new partner, and
women pregnant from the same partner as the first pregnancy, would show whether this hypothesis is correct. In chapter 4, not only memory T cell populations in the decidua had higher CD69+ expression in multigravid women compared to
primigra-vid women, also the majority of the other T cell subsets analysed had higher CD69+
proportions. It could be suggested that this is due to higher activated memory T cells in multigravid women compared to primigravid women, since memory T cells have been shown to induce activation of other T cells during an active immune response16.
Lower CD69 expression on T cells, as seen in preeclampsia, might have several consequences. It has been shown that CD69 expression on CD4+ T cells is
cru-cial for the formation of functional CD8+ memory T cells33, memory B cells34, and
long-lived plasma cells35,36. We showed increased CD69 expression on T cells and
memory T cells during normal human pregnancy in chapter 3 and chapter 4. But in
chapter 6 and chapter 7, lower CD69 expression on several memory T cell subsets in
preeclamptic women in the decidua and lower expression on all memory T cell subsets in the peripheral blood of preeclamptic women compared to healthy pregnant women was shown. With the knowledge of the role of CD69 in formation of immunologic memory, our findings could imply a less favorable environment for the formation of immunologic memory in preeclampsia. A reason for the lower CD69 expression in preeclampsia could be altered antigen presenting cell populations that have been found in preeclampsia, which might result in altered antigen presentation and with that altered activation of T cells in preeclampsia37,38. Interestingly, next to inducing
a pro-inflammatory response, immune regulatory functions of CD69 have also been suggested19,39. Previous studies showed CD69+ T cells secreting Tumor Growth
cells into Th1 and Th17 cells19,39,40. In light of CD69+ cells having immune regulatory
abilities, the lower CD69+ frequencies of memory T cells in the peripheral blood
and of several subsets in the decidua of preeclamptic women might have a role in the increased numbers of Th1 and Th17 cells in preeclampsia41,42. Lower CD69
fre-quencies in and after preeclamptic pregnancies in association with memory T cell formation, immune regulatory function, and preeclampsia recurrence risk could be focus for future studies.
Altered immune regulation has been found in preeclamptic patients in multiple studies43–45. Lower Treg cell proportions were reported in the peripheral blood and
the decidua in preeclamptic women43,44, however the memory cell subset within the
Treg cell population has not been studied much in pregnancy yet45,46. Treg memory
cells were found to have decreased suppressive capacity in preeclampsia46, but
were found present at higher proportions in peripheral blood of preeclamptic women compared to healthy pregnant women45. In chapter 7, we found lower CD4+ CM
Treg cell proportions in the decidua of late-onset preeclamptic women compared to healthy pregnancies. But we did not observe lower Treg memory cell proportions in other memory T cell subsets in decidual tissue of preeclamptic women compared to healthy women. In this chapter, the absence of substantial Treg memory cell alterations might be due to the gestational age of the different patient groups that were included. Treg cell proportions are known to fluctuate over the course of pregnancy47,48, and
the substantial, but insurmountable, average difference of 9 weeks in gestational age between early-onset preeclamptic and healthy decidual tissue at time of analysis in
chapter 7 might have affected our results.
In chapter 5, fetal sex dependent changes of the maternal T cell response were reported. In this chapter, we reported higher FOXP3 mRNA expression and higher interferon-γ (IFNγ) mRNA expression in first trimester decidual tissue of pregnancies with a female fetus compared to pregnancies with a male fetus. Previous studies also reported immune alterations with fetal sex, but the data in chapter 5 added additional value because immunologic data from the fetal-maternal interface in early human pregnancies with known pregnancy outcomes is unique, since nowadays no routine chorionic villous sampling is performed with advanced maternal age. This makes obtaining early pregnancy decidual tissue from ongoing pregnancies very rare. The transcription factor FOXP3 is expressed on Treg cells and is associated with immune regulatory function and fetal-maternal tolerance49–51. Since pregnancies with
a female fetus are associated with lower pregnancy complication risks compared to pregnancies with a male fetus52–55, it is proposed that fetal-maternal tolerance is
more sufficient in pregnancies with a female fetus4,13,56. The higher FOXP3 mRNA
expression found in chapter 5 would be in line with this hypothesis. In addition, even though demonstrated in a mouse study and not in humans, in chapter 9, altered immune adaptations in early pregnancy were associated with sex-specific effects on offspring. Although a causative link remains to be shown between the altered immune adaptations and the sex specific effects on offspring, the data in chapter 5 and
chapter 9 do emphasize the importance of the consideration of fetal sex in further
investigations in the field of pregnancy immunology and the delicacy of the immune balance in early pregnancy.
In chapter 5 we found higher IFNγ mRNA expression in decidual tissue of preg-nancies with a female fetus. Higher IFNγ mRNA expression has been associated with a pro-inflammatory state and pregnancy complications57, however many studies
claim that IFNγ also plays an important role in successful pregnancies, especially in early stages through contributions to spiral artery development and adequate placentation58–61. It should be noted that the higher IFNγ mRNA expression found in
chapter 5 was not found in a specific immune cell population but in decidual tissue
biopsies. Therefore, the higher IFNγ mRNA expression could be caused by immune cells such as Th1 cells and uterine NK cells which are known to be the main producers of IFNγ61–63. Higher IFNγ has been shown to induce proliferation of CD4+ cells into
Treg cells and some Treg cells have been shown to produce IFNγ which appeared critical for Treg immune regulatory function64–66. This might have contributed to the
higher FOXP3 in combination with higher IFNγ mRNA expression which we found in first trimester decidual tissue of uncomplicated pregnancies with a female fetus compared to a male fetus in chapter 5. Interestingly, in the mouse study in chapter 9, we found an increase of IFNγ expression in Treg cells and effector T cells from early pregnancy till mid gestation in the control group (placebo treatment). This increase was accompanied by an increase of the Treg cell population in this time frame. Even though mice and humans have a different placental anatomy, in both species mater-nal cells are in direct contact with fetal cells. Together with comparable immunologic homeostasis, mice are often used as a model to study immunology in pregnancy67.
From the mouse study in chapter 9 it seems that a balanced increase of IFNγ in early pregnancy is physiologic and therefore, as suggested by others58, IFNγ should not
be associated with unsuccessful reproduction prematurely, especially when found in early pregnancy.
Fluctuations of the immune responses in pregnancy are complex, balanced, spe-cifically timed, and above all, the exact fluctuations are incompletely understood. This makes that immune modulating therapies during pregnancy might interfere with physiologic fluctuations that we don’t know of and thereby immune modulation could be potentially dangerous for fetal and maternal health. As reviewed in chapter 8, different types of immune modulation therapies are proposed to improve insufficient maternal immune adaptations. The immune modulatory drug prednisolone is further investigated in a mouse model in chapter 9. Prednisolone is used to treat women with implantation failure, with the rational of suppressing a supposed abnormal immune response. However, in chapter 9, we showed that prednisolone disrupts normal maternal immune adaptations, with lower Treg cell frequencies and numbers in the para aortic uterus draining lymph nodes and effects on pregnancy outcomes and offspring development, with lower placental weights, lower body weights after birth and lower adrenal weights in male offspring. The suppressed immune response found after prednisolone treatment is in line with literature showing lower Treg cell numbers and lower IFNγ after prednisolone treatment in non-pregnant circumstances68–70.
This mouse model should be extrapolated to human subjects with caution. Since prednisolone is prescribed to women with infertility, implantation failure or recurrent miscarriage without knowing the exact pathophysiology, it is presumably prescribed to many women with physiologic immune adaptations to pregnancy. Since specific active immune processes, rather than broad immune suppression, are necessary for these physiologic immune adaptations in pregnancy and aspecific immune suppres-sion with prednisolone might interfere with these adaptations71,72, we attempted to
demonstrate the effects of prednisolone in healthy early pregnancies and did not include mice prone to implantation failure or miscarriage. Also, in chapter 9, we did not provide causal evidence that the immune disruptions caused by prednisolone are responsible for the altered pregnancy outcomes and offspring development. Investigations of the placenta after prednisolone administration could provide more answers regarding causality. Even though we did not provide causal evidence, the association of prednisolone treatment in early pregnancy with modulated pregnancy outcomes and offspring development do stress the importance of being cautious with immune modulatory therapies in early pregnancy. Therefore, with the current know-ledge, including the data presented in this thesis, we should reconsider the implication of immune modulatory treatments in reproduction. More studies, including studies powered for detecting abnormalities in postnatal development, should be done before application of treatments in clinical practice.
Future perspectives
Multiple studies on fetal-maternal tolerance investigated fetal-paternal T cell speci-ficity2,3,13,73–75, but remarkably, the majority of these studies are performed in mouse
models2,3,73. In mice, fetal-paternal antigen specificity can be investigated in several
ways; by adoptive transfer experiments, or ovalbumin transfected mice mating models. In this thesis, the T cell populations are studied without identifying the fetal-paternal antigen specific cell population. Only very limited number of human studies described memory T cells in pregnancy and therefore we aimed to first acquire knowledge on memory T cell fluctuations in healthy and preeclamptic pregnancies. The knowledge gained in this thesis suggests their involvement in fetal-maternal tolerance and the-rewith provides reasons to continue with functional analyses and antigen specific tests to further elucidate their role. In human studies determination of fetal-paternal antigen specific memory T cells is mainly achieved through investigating specificity for Y-chromosome encoded antigens in women pregnant with a male fetus using HY-dex-tramers13,74,75. But, the proportion of HY-specific T cells appears to be low relative to
the total memory T cell population present in the decidua or the peripheral blood13,76.
This is presumably due to the fact that the antigens encoded by the Y-chromosome only represent allogeneic antigens of 1 of the 23 allogeneic chromosomes, so the HY-spe-cific memory T cell population is not a complete representation of the fetal-paternal antigen specific memory T cell population. It remains complex to investigate antigen specificity of memory T cells in humans, so future studies should focus on a feasible approach. A suggested approach would be T cell receptor sequencing which will hopefully be broadly available in a matter of years. Complexity then comes with the wide variety of fetal-paternal antigens specific for each father. Identification of TCR specificity groups could be used to predict HLA-restriction and therewith fetal-paternal specificity77. Presumably, TCR sequencing techniques would greatly accelerate the
analysis of memory T cells in the field of reproductive immunology.
In this thesis, we present findings showing different numbers and activational status of memory T cells in pregnant women compared with non-pregnant women and in preeclamptic women compared with healthy pregnant women. Our results may suggest a beneficial role for memory T cells in fetal-maternal immune tolerance. Investigations of the functionality of the memory T cell subsets in the different conditions are neces-sary to confirm the suggested involvement. Limitations come with the low number of cells that can be harvested from the tissues at the fetal-maternal interface. Thus, future studies should optimize isolation techniques and functionality analyses, so that more cells could be isolated and functional tests are possible with lower cell numbers.
Besides, switching to rodent models should not always be the first alternative when human studies appear complex. Whilst realizing that animal models can not be entirely replaced, the current developments in organoid development78, tissue engineering,
and organ on a chip methods79,80, give a wealth of approaches for models replacing
animal studies, also in the reproductive field78,81. Recently, trophoblast organoids have
been developed78 and placenta on a chip devices have been invented79, which have
been shown to have similar functioning to in vivo tissues and therewith replacing some animal experiments. In addition, establishment of high quality and large biobanks of gestational tissue would be critical for taking the field of reproductive immunology a step closer to target immune mechanisms for treatment82. In this way multifactorial
diseases like preeclampsia can be better studied because homogenous groups with large sample sizes can be studied. Also, organoids could be developed of tissues of complicated pregnancies to further study cellular and molecular pathways of disease as has already been done succesfully for colorectal cancer83.
In chapter 9, we conclude that application of immune modulatory treatments might need more research before safely implementing these in clinical practice. However, making use of immunologic memory in a natural way could be an option of applying the current knowledge in reproductive immunology in a safer way. Previous studies associated exposure to seminal fluid with lower incidence of recurrent miscarriage and improved IVF outcomes84,85. The findings in this thesis are in accordance with
the hypothesis that immunologic memory might contribute to these lower pregnancy complication risks in a following pregnancy. Therefore, the findings in this thesis sup-port the suggestion that women with an increased risk of pregnancy complications might benefit from higher pre-conceptional exposure to paternal antigens. It could be proposed to develop medication containing paternal antigens in a high dose to boost immunologic memory formation - as an addition to more sexual contact with seminal fluid exposure - in women suffering from, or at high risk for, immune asso-ciated pregnancy disorders such as recurrent miscarriage or implantation failure. This way, the immune system is not modulated but rather boosted to elicit a better tolerating response in a subsequent pregnancy, possibly increasing the chances of pregnancy success.
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