Journal of Reproductive Immunology 108 (2015) 12–25

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Maternal microchimerism: lessons learned from murine models Ina Annelies Stelzer ∗ , Kristin Thiele, Maria Emilia Solano ∗ Laboratory of Experimental Feto-Maternal Medicine, University Medical Center Hamburg-Eppendorf, Germany

a r t i c l e

i n f o

Article history: Received 9 September 2014 Received in revised form 4 December 2014 Accepted 14 December 2014 Keywords: Maternal microchimerism Mouse pregnancy Fetal immune ontogeny Feto-maternal immune cross-talk In utero programming

a b s t r a c t The presence of maternal cells in the organs of the offspring is referred to as maternal microchimerism (MMc). MMc is physiologically acquired during pregnancy and lactation and can persist until adulthood. The detection of MMc in a variety of human diseases has raised interest in the short- and long-term functional consequences for the offspring. Owing to limited availability and access to human tissue, mouse models have become an essential tool in elucidating the functional role of MMc. This review compiles the detection techniques and experimental settings used in murine MMc research. It aims to summarize the potential mechanisms of migration of MMc, pre- and postnatal tissue distribution, phenotype and concatenated function, as well as factors modulating its occurrence. In this context, we propose MMc to be a materno-fetal messenger with the capacity to critically shape the development of the offspring’s immunity. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Why are we interested in maternal microchimerism? Maternal microchimerism (MMc) is defined as the presence of maternal cells in the organs of the fetus and offspring. MMc cells can be acquired during pregnancy and lactation and persist until adulthood. The variety of MMc cell phenotypes, its widespread location in the offspring’s organism, and its detection in the context of human auto-immune diseases have raised interest in its short- and long-term functional role for the offspring. To date, the importance of MMc in humans has been discussed in a wealth of settings, such as tolerance induction or priming of the child’s immune response.

∗ Corresponding authors at: Laboratory of Experimental Feto-Maternal Medicine, Department of Obstetrics and Fetal Medicine, University Medical Center Hamburg-Eppendorf, Martinistr. 52 – 20246 Hamburg, Germany. Tel.: +49 40 7410 58710; fax: +49 40 7410 52395. E-mail addresses: [email protected] (I.A. Stelzer), [email protected] (M.E. Solano). http://dx.doi.org/10.1016/j.jri.2014.12.007 0165-0378/© 2015 Elsevier Ireland Ltd. All rights reserved.

Microchimerism-induced tolerance is evident in allograft transplantation, as recipients transplanted with maternal tissue experienced a decreased acute rejection rate, graft failure after 6 months, and graft-versus-host disease (GVHD) in comparison to those transplanted with paternal tissue (Joo et al., 2013; Nijagal et al., 2012; Van Rood et al., 2002). This tolerogenic effect has been attributed to the child’s exposure to non-inherited maternal antigens (NIMA) during pregnancy (Dutta and Burlingham, 2011b). On the contrary, higher prevalence or frequencies of MMc were associated with various auto-immune diseases (Artlett et al., 2001; Lambert et al., 2004; Nelson et al., 2007; Reed et al., 2004; Vanzyl et al., 2010; Ye et al., 2014b). This suggests that the priming of the developing offspring immunity by foreign MMc cells could contribute to the origins of auto-immunity (Leveque and Khosrotehrani, 2011). However, MMc cells do not appear to be the effectors mediating the inflammatory process and direct evidence of their role in the onset and progression of auto-immune responses remains elusive.

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The clinical relevance of human MMc (Jeanty et al., 2014; Nelson, 2012) and its functional role in the context of transplantation (Dutta and Burlingham, 2011b; Eikmans et al., 2014) or auto-immunity (Leveque and Khosrotehrani, 2011; Ye et al., 2014a) have been recently reviewed. Despite the growing body of evidence, research into MMc is restricted by the limited access to human fetal and adult organ samples, rendering animal models an essential tool for research. The aim of the present review is to provide a comprehensive overview of the state of the art in MMc research carried out in mouse models. Considering publications dating back to the 1990s, experimental settings and techniques used to detect MMc and potential mechanisms of migration into the host are summarized. The tissue distribution, phenotype, and function of microchimeric cells, in addition to factors modulating their occurrence in the host’s tissues are summarized. In this context, evidence supporting the role of MMc as a maternal messenger involved in shaping the fetal and postnatal developing immunity is discussed. 2. How are MMc cells identified? In humans, MMc cells are differentiated using the human leukocyte antigen (HLA) haplotype, which differs between mother and child. The HLA haplotype is identified by different polymerase chain reaction (PCR)-based methodologies or fluorescence-activated cell sorting. Alternatively, sex chromosome-based fluorescence in situ hybridization (FISH) has been used to identify single maternal cells in offspring’s tissues (Eikmans et al., 2014). Immunofluorescence- and PCR-based approaches can be combined to verify the genetic identity of single microchimeric cells (Kroneis et al., 2010). In contrast, mouse models offer a broader spectrum of markers and methods that permit the detection of a low amount of MMc cells among a high quantity of offspring’s cells. During the last 20 years, various mouse models have been designed to generate detectable genetic differences between mother and fetus. For example, this can be achieved by the transfer of blastocysts into a genetically different pseudo-pregnant dam. Initially, mouse strains that differ in the H-2 genes, which comprise the major histocompatibility complex class I (MHC I) homologous to HLA in humans (Marleau et al., 2003; Piotrowski and Croy, 1996; Shimamura et al., 1994), were employed. The posterior application of foster mothers that express reporter transgenes such as LacZ or enhanced green fluorescent protein (eGFP) facilitated the detection of MMc in the offspring (Marleau et al., 2003; Piotrowski and Croy, 1996; Unno et al., 2010). Following a similar approach, other models consisted of the adoptive transfer of cells, i.e., from human origin or labeled radioactively (Chen et al., 2008; Wienecke et al., 2012), into the pregnant female. When tracking radioactively labeled cells, it may be taken into account that radioactive traces on cellular debris could also be exchanged via the placenta and contribute to the signal present in the fetuses. Despite enabling the detection of MMc cells in the fetus, these experimental approaches represent a significant intervention during pregnancy, which could affect the

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MMc per se. To overcome this limitation, different mating combinations employing mice that differ in the H-2 genes, in the congenic common leukocyte marker CD45 subtypes (CD45.1, CD45.2), or in the expression of transgenic markers (i.e., eGFP) allow homozygous maternal cells to be detected among heterozygous offspring cells in undisturbed pregnancies. An overview of the most relevant markers and mating models is provided in Table 1. The experimental model selected must be accompanied by an appropriate detection method. When working with offspring in the early stages of life (fetus or neonates), the size of the organs represent a relevant limitation. Hence, the detection of MMc cells is not only dependent on the technical approach employed, but also on the amount of cells or tissue that can be obtained at a certain age. The methods more frequently used in the context of MMc are microscopy-based methodologies, flow cytometry, and PCR. Microscopy-based methodologies include fluorescence microscopy or immunohistochemistry, which provide information on the MMc cell morphology and tissue localization. As an example, CD1 fetuses obtained from blastocyst transfer into LacZ-transgenic dams showed positive signals following X-Gal staining (Piotrowski and Croy, 1996). EGFP is a suitable marker for optical detection methods as it possesses self-catalyzed chromophore formation and appears green under excitation light (Okabe et al., 1997). However, compared with PCR-based methods or flow cytometry, microscopy-based methodologies provide mostly semi-quantitative information, which often relies on the selected sections, visual fields or areas analyzed. Flow cytometry allows not only the identification, but also the phenotypical characterization of MMc cells. However, this MMc cell detection may be affected by cell auto-fluorescence, unspecific antibody binding and spectral overlap in the fluorescent signal emission. EGFP and CD45 have been recognized to be pivotal markers in initial MMc detection. The fluorescence of eGFP requires no other cofactors for its detection, as its major excitation peak matches the spectral characteristics of the commonly used fluorescein isothiocyanate (FITC) (Chalfie et al., 1994; Inouye and Tsuji, 1994), whereas CD45.1 and CD45.2 can be reliably discriminated using specific antibodies. A comparative sensitivity analysis of our group revealed equal detection limits for CD45 and eGFP (Thiele et al., 2014). In order to improve the flow cytometric detection of naturally occurring hematopoietic MMc cells among haploidentical fetal cells, we recently introduced a mouse model combining CD45.1, CD45.2, and H-2 haplotypes as independent markers (Solano et al., 2014). Here, we aimed to further characterize the MMc cells and simultaneously minimize the false-positive signals that may occur because of the intrinsic technical limitations as discussed above. Finally, quantitative PCR (qPCR) can be employed to genetically detect and quantify the occurrence of MMc cells in the organs of the offspring. To date, established protocols allow discrimination of the H-2 haplotype and the presence of the eGFP gene. In contrast, a molecular approach to identifying the CD45 subtype is not yet available, as CD45.1 and CD45.2 differ in only 12 nucleotides (Zebedee et al., 1991). The limitation of a DNA-based method is, however, that

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Table 1 Experimental settings and techniques used to detect maternal microchimerism in fetal and adult mouse tissue. Marker

LacZ+ /Rosa26

eGFP+

Mating model

Technique

Location

♀

♂

–Geneic

ID

Setup

Thy

Liver

Spl

BM

Lung

Heart

LacZ tg ROSA26

Allo

×

Histochemistry Flow cytometry Histochemistry

× × —

× × —

× × —

×

×

×

Piotrowski and Croy (1996)

LacZ tg ROSA26

Outbred CD1

Allo

Blastocyst transfer Blastocyst transfer Blastocyst transfer

×

LacZ tg ROSA26

Outbred CD1-scid/scid Outbred CD1

Histochemistry PCR

×

×

× ×

× ×

Marleau et al. (2003)

Heterozygous GFP (+/−)

GFP(−/−) (C57Bl6)

Syn

NI

×

×

×

×

NI

Fluorescence microscopy Flow cytometry Histochemistry Nested PCR

×

×

Allo Outbred Syn

NI

Fluorescence microscopy qPCR

Syn

NI

Allo

GFP(−/−) (C57Bl6) FvB (H-2q) Outbred ICR GFP(−/−) (C57Bl6) CCL3−/−GFP−/− (C57Bl6) GFP(−/−) (Balb/c)

Syn

Allo

NI

Heterozygous GFP (+/−)

GFP(−/−) (C57Bl6)

Syn

NI

Fluorescence microscopy Fluorescence microscopy qPCR qPCR

C57Bl6 wildtype CD45.2 RAG2−/− CD45.1+/−

C57Bl6 congenic CD45.1 RAG2−/− (C57Bl6)

Syn

NI

Flow cytometry

—

NI

Flow cytometry

×

Luciferase

luciferase transgene-tg FVB

Wildtype FVB

Syn

NI

qPCR

MHC class I H-2d

Foster mother, pseudo-pregnant (Balb/c) C57Bl6

AKR

Allo

Blastocyst transfer

Flow cytometry

Balb/c

Allo

NI

PCR

B6D2F1 (H-2b/d)

C57Bl6 (H-2b/b)

Allo

NI

Nested PCR

×

B6D2F1 (H-2b/d)

C57Bl6 (H-2b/b)

Allo

NI

qPCR

×

B6D2F1 (H-2b/d)

C57Bl6 (H-2b/b)

Allo

NI

qPCR

F1 (B10.D2xB10) (H-2b/d)

B10.BR (H-2d)

Syn

NI

Nested PCR

Heterozygous GFP (+/−) CCL3+/−GFP+/− C57Bl6 Heterozygous GFP (+/−)

Syn

×

Blood

Kid

Brain

LN

Plac

Zhou et al. (2000) × × ×

× ×

× ×

—

×

—

×

×

×

×

×

×

×

×

×

× ×

×

Vernochet et al. (2005, 2007) Dutta et al. (2009) Unno et al. (2010) Lopez-Guisa et al. (2011)

× ×

—

—

×

×

— ×

×

Dutta and Burlingham (2011a)

×

Nijagal et al. (2011) Roy et al. (2011)

×

×

×

×

×

×

×

—

×

Shimamura et al. (1994)

×

Wan et al. (2002) Andrassy et al. (2003) Dutta et al. (2009) Dutta and Burlingham (2010) Araki et al. (2010)

× × ×

×

Su et al. (2008)

× × ×

×

×

×

×

×

×

×

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Heterozygous GFP (+/−)

CD45.1+ /CD45.2+

Reference

Arvola et al. (2000) ×

No organ distinction

ELISA Syn B cell deficient ␮ (−/−) B cell deficient ␮ (+/−)

Cr

IgG

C57Bl6 (CD45.1)

Syn

×

NI

Radioactive labeling Adoptive transfer

PCR NI BALB/c

IL-2/GFP promotor (BALB/c) C57Bl6 (CD45.1) 51

NeoR+

IL-2

Syn

Syn C57Bl6 neoR(−/−)

Allo C57Bl/6

Foster mother, pseudo-pregnant (Balb/c) C57Bl6 neoR(+/−) MHC class II

DBA/2 (H-2Dd/d) TEa tg C57Bl6 (H-2Db/b) C57Bl6 (H-2Db/b)

Allo C57Bl6 (H-2b/b) B6D2F1 (H-2b/d)

Abbreviations: AKR: inbred strain with H2 haplotype k; Allo: allogeneic; B6D2F1: F1(C57Bl6xDBA/2); B10: C57Bl10; B10.D2: B10.D2/n Sn Slc; B10.BR: B10.BR/Sg Sn Slc; Cr51: sodium chromate; eGFP: enhanced green fluorescent protein; ID: immunodeficiency; IgG: Immunoglobulin G; IL: interleukin; Kid: kidney; LN: lymph node; MHC: major histocompatibility complex; neoR: neomycin resistance gene; NI: no intervention; PCR: polymerase chain reaction; qPCR: quantitative PCR; Plac: placenta; Spl: spleen; Syn: syngeneic; tg: tagged; Thy: thymus; × = detected; — = not detected, empty: not analyzed.

× ×

×

× × NI

qPCR

—

× ×

— PCR

Flow cytometry NI Allo

Blastocyst transfer

× × Flow cytometry NI Allo

NI

qPCR

×

×

×

×

×

—

×

×

×

Kaplan and Land (2005) Wrenshall et al. (2007) Wienecke et al. (2012)

Shimamura et al. (1994)

Dutta and Burlingham (2011a) Leveque et al. (2014)

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target detection, although quantified as genomic equivalents per fetal cells, does not allow phenotypical or functional characterization of MMc. Future endeavors in experimental MMc studies may rely on single-cell analysis, which allows the comprehensive characterization of the MMc cell transcriptome, as has been performed in fetal microchimerism research in humans. 3. How do the MMc cells reach the fetus? 3.1. Maternal cells cross the placenta by transendothelial migration Although microchimerism is assumed to originate from transplacental trafficking, the mechanisms by which cells cross the feto-maternal interface in the mouse and in humans are unclear. During the establishment of blood circulation in the murine placenta at mid-gestation, maternal cells progressively invade the labyrinthine zone starting at gestation day (gd) 13 to reach its maximum between gd 17 and 19 (Vernochet et al., 2007). These cells have been observed to spread across the zone of materno-fetal blood exchange, which could facilitate the traffic through the placenta (Unno et al., 2010, Vernochet et al., 2007). In order to reach the fetal circulation, maternal cells in the blood spaces of the placental labyrinth have to cross the mononuclear trophoblast cell layer, two layers of multinucleated syncytiotrophoblast, and, after traveling the labyrinthine stroma, migrate through the basal membrane and fetal endothelial cell layer (Rossant and Cross, 2001) in a process of transendothelial migration. It has been hypothesized that the process of cell adhesion and transmigration of lymphocytes in the placenta is similar to that seen at the blood–brain barrier (Dawe et al., 2007). The feto-maternal interface appears to express the necessary molecular machinery, including molecules for cell capture, adhesion, and transmigration. Whilst in humans the expression of cellular adhesion molecules that could mediate cell traffic has been addressed by several studies (Cartwright and Balarajah, 2005; Dye et al., 2001; Xiao et al., 1997), in mice it has been less frequently investigated. For early rolling, a unique E-selectin gene expression pattern has been attributed to the trophoblast (Milstone et al., 2000), which also expresses P-selectin (Fernekorn et al., 2007). Junctional adhesion molecules may play a role in facilitating integrin-mediated MMc cell transendothelial migration. Among them, PECAM-1 (Looman et al., 2007; Prados et al., 2011), VE-cadherin, and ␤-catenin (Rutland et al., 2007) are expressed by endothelial cells at the murine feto-maternal interface. The presence of the recently discovered CD99(L2), a novel player in leukocyte recruitment and extravasation (Seelige et al., 2013), remains to be histologically determined in the mouse placenta. Among the superfamily of junctional adhesion molecules (JAMs), JAM2 is expressed in early murine gestation (Su et al., 2012). Interestingly, JAM2 is expressed in human placenta (Aurrand-Lions et al., 2002; Johnson-Leger et al., 2002) and its ligand, the integrin VLA-4, has been shown to be necessary for transplacental migration (Chen et al., 2008). This makes JAM2 a candidate protein that could be involved

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in MMc transmigrational processes at the murine fetomaternal interface. It is possible that the selective passage of MMc cells is driven by gradients of chemo-attractants existing across fetal and maternal compartments. In human pregnancies, the vascular endothelial growth factor (VEGF)-A forms a gradient that increases in concentration from the maternal blood plasma to the human umbilical cord blood. In the rat and in humans VEGF-A has been shown to promote cell migration via a mechanism that involves VEGFR-1, and the integrins ␣4␤1 (VLA-4), ␣2␤1, and ␣5␤1 (Chen et al., 2008). It remains unknown whether this mechanism is also of relevance in mice. The only available study on MMc cell trafficking in mice revealed that the chemokine ligand (CCL) 3, a chemo-attractant highly expressed by the fetal trophoblast during late gestation, is not essential for MMc cell migration (Unno et al., 2010). Cell migration through the placental into the fetal circulation seems to involve multiple mechanisms, as is seen in other migration processes. Regulatory machinery may enable a selective passage of cells, as the phenotype of the MMc cells in the fetal circulation differs from the maternal peripheral blood composition (Nijagal et al., 2011). 3.2. Lactation as a source of persistent MMc Interestingly, transplacental trafficking does not appear to be the only source of MMc. Different approaches suggest that post-partum nursing might be relevant for the persistence of maternal semi-allogeneic cells in the offspring’s organs until adulthood. MMc was transiently present postnatally, when passaged milk leukocytes were found 3 and 6 days after birth in the neonatal liver (Zhou et al., 2000). Similarly, Dutta et al. (2009) showed that breast-feeding alone was sufficient to induce MMc in the liver of foster-nursed offspring. Nursing B-cell-deficient neonates by wild-type foster dams resulted in IgG-secreting cells still detectable in the adult offspring, demonstrating that B cells had been transferred postnatally via the milk (Arvola et al., 2000). Oral exposure to NIMA is claimed to be essential for the persistence of MMc in the offspring (Dutta and Burlingham, 2010; Dutta et al., 2009). In bone marrow transplantation, in utero and oral exposure to NIMA was required for the maximal reduction of GVHD, while oral exposure to NIMA by breast-feeding alone was already sufficient to reduce GVHD (Matsuoka et al., 2006). Similarly, heart allografts expressing NIMA were rejected more readily by offspring that had only been in contact with the maternal antigen in utero, compared with both in utero and orally exposed offspring (Andrassy et al., 2003). Tolerance induction to NIMA even failed when the offspring was not exposed to maternal milk (Andrassy et al., 2003; Dutta et al., 2009). Apparently, lactation seems not only to be a second source of maternal cell transfer, but might also be a route to boosting tolerance toward NIMA-expressing MMc cells, as the offspring’s early postnatal immune system is inclined to develop cytotoxic immune responses (Opiela et al., 2008). How maternal cells manage to escape acidity and digestion in the gastrointestinal system and enter the blood circulation of neonates, however, is not clear (Dutta and Burlingham, 2011b). Lactational transfer could thus play

a significant role in the long-term maintenance of MMc and could contribute to an explanation of the variability of MMc levels among humans, who may differ in their nursing history. 4. When can MMc cells be detected and where are they located? As observed in humans, murine MMc cells can be detected in multiple fetal, neonatal, and adult organs, such as peripheral blood, lymphoid organs, but also nonlymphoid tissues. In Table 2, we summarize the current evidence for the tissue distribution and phenotype of MMc found in prenatal and postnatal mice, including corresponding references to research in human MMc. Owing to the variety of experimental set-ups, technical methods used, and organs analyzed, the detection of MMc provides sometimes contradictory results in different studies, which are displayed in Table 1. There is compelling evidence that in mice MMc originates as early as mid-gestation upon the establishment of the blood circulation in the hemochorial placenta (Marleau et al., 2003; Rossant and Cross, 2001). From gd 12.5, MMc cells are found in the fetal thymus, although only in immune-deficient offspring, and in peripheral blood. On gd 13.5 MMc can also be detected in the liver, which is the major hematopoietic organ at that gestational age (Vernochet et al., 2005). In parallel with the rapid build-up of the prenatal immune system, MMc appears to become a widespread phenomenon in late murine pregnancy. Although Marleau et al. (2003) did not find any MMc cells present in whole fetal tissues until gd 15.5, on gd 16.5 they observed abundant MMc in bone marrow and less prominently in the liver. Increased levels of radioactivity accumulated in gd 19 fetuses compared with gd 15 upon adoptive transfer of radioactive cells into the maternal circulation (Wienecke et al., 2012). In contrast, the frequency of MMc cells among fetal blood cells decreased from gd 13.5–14.5 to late gestation and neonatal life (Nijagal et al., 2011). This relative decrease could be explained by increased fetal blood cellularity due to the progressing hematopoiesis or by the emigration of MMc cells into the fetal organs. Indeed, in late gestation, MMc cells were more prevalently found in the liver, bone marrow, and spleen, followed by the thymus and also the lung (Piotrowski and Croy, 1996; Unno et al., 2010; Zhou et al., 2000). In neonatal life, MMc has a similar distribution to that observed during late gestation. Still, in the early postnatal period, regulation of the MMc level is apparent. MMc cell frequency was observed to increase from postnatal day (pnd) 1, peak at pnd 7 and then, via a nadir on pnd 14, recover on pnd 21 the levels observed on pnd 1 (Su et al., 2008). This may reflect the dynamic transition of the prenatal immune system, which is more tolerogenic toward MMc, to the adult immunogenic program (Burt, 2013; Dakic et al., 2004), which could be accompanied by the deletion of semi-allogeneic MMc cells. The hematopoietic organs containing maternal cells were shown to continually increase until young adulthood (Vernochet et al., 2005). In young adults, maternal cells are commonly found in

Table 2 Age-dependent location and phenotype of maternal microchimeric cells in mouse prenatal, neonatal and young adult tissue.

Prenatal

Tissue

Mid-gestation (gd 12.5–15.5)–late gestation (gd 15.5-parturition)

MMc cell phenotype

Reference (mouse)

Reference (corresponding human evidence for respective tissue and age)

Thymus

12.5 (immune-deficient)

Isolated cell cluster in thymic region T, B, NK cells ND Lymphoid and myeloid cells T cells T, B, NK cells m: Isolated scattering of cells m: Expanding cords of cells ND Lymphoid and myeloid cells T, B, NK cells m: Lymphocyte-like cells lining hematopoietic regions in BM cavities m in long bones: Isolated cluster of few cells m in skeleton and long bones: Expanding cords of cells m: Cells expand into hematopoietic regions in BM cavities ND m: Isolated cluster of few cells lymphoid and myeloid cells

Piotrowski and Croy (1996)

Jonsson et al. (2008)

Liver

18.5 (immune-deficient) 18.5 18.5–19.5

Spleen

Lymph nodes Peripheral blood

Placenta labyrinth

Lung Heart Complete fetus

Kidney, adrenal gland, pancreas, brain, ovary, testis

15.5-parturition 18.5 (immune-deficient) 18.5 12.5–15.5 18.5–19.5 13.5 17.5–19.5 18.5 17.5–19.5 18.5 18.5 (immune-deficient) 18.5 (immune-deficient) 14.5 17.5–19.5

T, B, NK, dendritic cells, macrophages, granulocytes Granulocytes, T, B cells ND ND ND Effector Th cells (adoptively transferred) ND m: Isolated cluster of few cells ND ND Effector Th cells (adoptively transferred)

Vernochet et al. (2005) Piotrowski and Croy (1996) Zhou et al. (2000) Vernochet et al. (2005) Vernochet et al. (2005) Marleau et al. (2003) Piotrowski and Croy (1996) Unno et al. (2010) Zhou et al. (2000) Vernochet et al. (2005) Marleau et al. (2003)

Jonsson et al. (2008)

Piotrowski and Croy (1996) Piotrowski and Croy (1996) Marleau et al. (2003)

Vernochet et al. (2005) Piotrowski and Croy (1996) Zhou et al. (2000) NA Nijagal et al. (2011) Nijagal et al. (2011) Vernochet et al. (2007) Vernochet et al. (2007) Unno et al. (2010) Wienecke et al. (2012)

Jonsson et al. (2008)

Mold et al. (2008) Lo et al. (1998), Petit et al. (1997)

Jonsson et al. (2008)

Unno et al. (2010) Piotrowski and Croy (1996) Piotrowski and Croy (1996) Zhou et al. (2000) Wienecke et al. (2012)

Jonsson et al. (2008)

NA

Jonsson et al. (2008)

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Bone marrow

15.5-parturition 18.5 (immune-deficient) 18.5 13.5 15.5-parturition 16.5 18.5 (immune-deficient) 18.5 18.5 15.5-parturition 16.5

Jonsson et al. (2008)

17

18

Table 2 (Continued)

Postnatal - neonate

Neonate–young offspring (pnd1–pnw3)

MMc cell phenotype

Reference (mouse)

Reference (corresponding human evidence for respective tissue and age)

Thymus

Neonate Pnw3 Pnd0-45 Neonate Pnd5

Leveque et al. (2014) Vernochet et al. (2005) Kaplan and Land (2005) Leveque et al. (2014) Zhou et al. (2000)

Srivatsa et al. (2003)

Pnd2 Pnw3 Pnd1 Pnd2 Pnw2 Pnw3 Pnd0-45 Pnd1-21 Pnd0-45 Pnd1-21 Pnd1-21 Neonate

T cells (transgenic) T, B, NK cells ND T cells (transgenic) Derived from lactational transfer m: Cluster of cells T, B, NK cells ND ND ND ND ND ND ND ND ND T cells (transgenic)

Neonate Pnd1-21 Pnd1-21

T cells (transgenic) ND ND

Leveque et al. (2014) Su et al. (2008) Su et al. (2008) NA

Liver

Bone marrow Spleen

Brain Lung Heart Skin

Small intestine Kidney Peripheral blood

Thyroid gland, pancreas, tonsils, adenoids, bladder

Zhou et al. (2000) Vernochet et al. (2005) Shimamura et al. (1994) Zhou et al. (2000) Marleau et al. (2003) Vernochet et al. (2005) Kaplan and Land (2005) Su et al. (2008) Kaplan and Land (2005) Su et al. (2008) Su et al. (2008) Leveque et al. (2014)

NA

Srivatsa et al. (2003), Stevens et al. (2009), Stevens et al. (2003)

Srivatsa et al. (2003), Stevens et al. (2009)

Stevens et al. (2009) Stevens et al. (2003) Srivatsa et al. (2003), Stevens et al. (2009), Khosrotehrani et al. (2006)

Stevens et al. (2009) Cord blood: Mold et al. (2008), Hall et al. (1995), Saadai et al. (2012) Children and adolescents: Artlett et al. (2001), Kowalzick et al. (2005), Maloney et al. (1999), Nelson et al. (2007), Reed et al. (2004), Thompson et al. (2013) Jonsson et al. (2010), Srivatsa et al. (2003), Stevens et al. (2009), Stevens et al. (2003)

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Tissue

Postnatal - young adult Thymus

Bone marrow

Pnw6, 10–12 Pnw4 Pnw6 Pnw6 (immune-deficient) Pnw6, 10–12 Pnw6-8

T, B, NK cells epithelial cells T cells (transgenic) T, B, NK cells Among lin+ cells, hematopoietic stem cells, myeloid cells, mesenchymal stem cells ND T cells (adoptively transferred) T, B, NK cells T, B cells ND Among antigen-presenting cells, myeloid cells, T cells

Vernochet et al. (2005) Dutta et al. (2009) Leveque et al. (2014) Vernochet et al. (2005) Dutta et al. (2009), Dutta and Burlingham (2010)

Marleau et al. (2003) Wan et al. (2002) Vernochet et al. (2005) Roy et al. (2011) Marleau et al. (2003) Dutta et al. (2009), Dutta and Burlingham (2010), Dutta and Burlingham (2011a) Leveque et al. (2014) Dutta et al. (2009) Vernochet et al. (2005) Dutta et al. (2009) Araki et al. (2010) Dutta and Burlingham (2011a) Dutta et al. (2009)

Adult Pnw6-8 Pnw6 Pnw6-8 Pnw8-12 Adult Pnw6-8

T cells (transgenic) ND T, B, NK cells ND ND ND ND

Heart

Pnw6-8 Pnw4 Pnw6-8 Adult Pnw6-8

Dutta et al. (2009) Wan et al. (2002) Dutta et al. (2009) Dutta and Burlingham (2011a) Dutta et al. (2009), Dutta and Burlingham (2010)

Small intestine

Adult Adult

ND ND ND ND Among lin+ cells, cardiac tissue macrophages, cardiomyocytes, endothelium, smooth muscle cells, cardiac stem cells ND T cells (transgenic)

Kidney

Pnw7-11

Hematopoietic cells, proximal tubular cells, endothelial cells

Lopez-Guisa et al. (2011)

Lymph nodes Peripheral blood

Liver Brain Lung

Pancreas

Dutta and Burlingham (2011a) Leveque et al. (2014)

NA

Lambert et al. (2004)

Lambert et al. (2004)

Kanold et al. (2013), Lambert et al. (2004), Loubiere et al. (2006), Sunku Cuddapah et al. (2010) Lambert et al. (2004); bile duct: Kobayashi et al. (2007) Lambert et al. (2004)

Lambert et al. (2004)

Suskind et al. (2011), Kiefer et al. (2012), Lambert et al. (2004)

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Spleen

Pnw6 Pnw6-8 Adult Pnw6 Pnw6-8

Lambert et al. (2004), Nelson et al. (2007), Vanzyl et al. (2010), Ye et al. (2014b)

Abbreviations: BM: bone marrow; gd: gestational day; lin: hematopoietic lineage marker; m: morphology; MMc: maternal microchimeric; ND: not determined; NA: not assessed in mouse; pnd: postnatal day; pnw: postnatal week.

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the bone marrow, thymus (Vernochet et al., 2005), and in blood (Araki et al., 2010) and lymph nodes (Dutta et al., 2009). Even up to 12 weeks of age, MMc is present in the bone marrow and at variable levels in the spleen of the offspring (Marleau et al., 2003). In non-lymphoid organs such as the brain, heart, skin, small intestine and kidney, MMc is more evident in early postnatal life, when it is also more frequently analyzed in these organs (Leveque et al., 2014; Su et al., 2008). Still, in neonatal and young offspring (pnd 0–45), the level of MMc in the brain correlated with the level of MMc found in lymphoid organs (Kaplan and Land, 2005) and the heart, liver, lung, brain, small intestine, and kidney remained sites of MMc in young adults (Dutta et al., 2009). This indicates a widespread peripheral distribution of MMc that is not restricted to the lymphoid compartment. At 6–8 weeks of age, though, the frequency of MMc per organ was decreased compared with pnd 1 offspring (Dutta et al., 2009). This could suggest an association between the persistence of MMc and the maturation of the offspring’s immune system. Taken together, during prenatal and early postnatal life, MMc takes place in the liver, bone marrow, thymus, and later in the spleen (Godin et al., 1995), at a time at which these organs are prominent sites of immune ontogeny. Semi-allogeneic MMc cells express NIMA, which are foreign to the offspring’s immune system. Hence, the presence of semi-allogeneic MMc could play a role in shaping the offspring’s immune ontogeny. Indeed, during late gestation, CD8+ T cell tolerance toward NIMA was shown to be achieved in the thymus (Akiyama et al., 2011), an organ of persistent MMc (Dutta et al., 2009; Leveque et al., 2014; Vernochet et al., 2005). In the periphery, gestational NIMA exposure induces the activation and expansion of specific CD4+ CD25+ FoxP3+ regulatory T (Treg) cells in young adults (Akiyama et al., 2011; Matsuoka et al., 2006; Molitor-Dart et al., 2007). Here, Tregs possessed a NIMA-specific suppressive function (Dutta et al., 2009; Matsuoka et al., 2006; Molitor-Dart et al., 2007), which correlated with the level of MMc found in immune and non-lymphoid tissues of the animals analyzed (Dutta et al., 2009). MMc appears to introduce antigens to the fetus, which can result in long-lasting tolerance towards these antigens. Thus, MMc may not only display maternal allo-antigens to the fetus, but also expose it to foreign material such as food and bacterial antigens in preparation for postnatal immune challenges (Mold et al., 2008). To date, the detailed mechanisms mediating this tolerance induction are unknown. We could speculate that similar to Treg induction, MMc may have an impact on other pillars of the innate and adaptive immune system, i.e., by influencing immune development in the liver, spleen, and bone marrow. During late gestation, B cells highly reactive to NIMA were partially deleted or entered anergy in the fetal liver and spleen (Vernochet et al., 2005). In contrast, shortly after birth, B cell development was skewed toward reactivity in bone marrow and spleen (Vernochet et al., 2005) upon encountering low levels of NIMA, and the splenic T cell response underwent longterm immunological priming (Opiela et al., 2008). These opposing peripartum responses toward foreign antigens demonstrate that the effect of MMc cells on the offspring’s

immunity is crucially dependent on the time and possibly location of occurrence. In summary, the widespread distribution of MMc would suggest that maternal cells directly influence the development of the offspring’s immune system, acting as a messenger that connects both immune systems. The observation of MMc in somatic tissue is remarkable, but the possible implication for the offspring’s physiology has been less thoroughly investigated. In order to address the potential role of MMc in the progeny it is necessary to consider the phenotype of MMc cells. 5. What is the phenotype of the MMc cells? Evidence for the transfer of MMc via transplacental migration from the maternal circulation (Vernochet et al., 2007) implies that MMc cells belong to the maternal blood pool, and hence most likely to the hematopoietic lineage. It is noteworthy that the MMc cell phenotype differs among the host organs (Table 2), suggesting that MMc cells are either of heterogeneous nature and/or have the capacity to differentiate locally. As early as gd 12.5–15.5, MMc cells from most hematopoietic cell lineages can be found in fetal lymphoid organs (Nijagal et al., 2011; Vernochet et al., 2005), where they can also be observed later in gestation and in postnatal life. Among the hematopoietic cells, T cells were the object of several studies (Leveque et al., 2014; Roy et al., 2011; Vernochet et al., 2005; Wan et al., 2002) owing to their relative longevity, capacity to specifically recognize non-self-antigens, and the central role they play in adaptive immune responses. MMc T cells were found in most fetal and neonatal lymphoid organs and endured until adult life, when both CD4+ and CD8+ T cells could be identified (Dutta and Burlingham, 2011a; Dutta et al., 2009). Interestingly, MMc CD4+ T cells were also found in non-lymphoid organs, such as the skin and small intestine of neonates and also in the small intestine of adult animals (Leveque et al., 2014). Whilst the functionality of these cells was not directly assessed, MMc T cells significantly influenced the offspring’s immunity, triggering either tolerance to foreign antigens (Fujii and Yamaguchi, 1992; Wan et al., 2002) (Box 1) or the loss of tolerance and consequent inflammation when MMc cells were highly allo-reactive toward the offspring’s antigens (Leveque et al., 2014; Roy et al., 2011) (Box 2). These contrasting effects leading to either tolerance induction or breakdown could also be influenced by age-dependent changes in the regulatory cell compartment in response to the MMc T cells. Interestingly, it has been shown that highly allo-reactive MMc T cells induced an increased frequency of Tregs in young offspring in the first place, whereas lower Tregs and higher effector T cell frequencies were observed in adult offspring (Leveque et al., 2014). In addition to allo-antigen presentation, MMc may contribute to the protection of the organism from foreign challenges during early life. For example, in a mouse model of in utero hematopoietic cell transplantation (IUHCTx) the engraftment was limited by MMc T cells (Nijagal et al., 2011). Furthermore, maternal antigens were also detected among CD11c+ and MCHII+ cell populations, indicating that

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Box 1: Induction of tolerance toward nonself-antigens – an advantage for transplant engraftment and allergy prevention? The observed advantage of NIMA exposure with regard to transplantation in humans is supported by studies in a mouse model that employs MHC class I H-2b/b offspring that is exposed to non-inherited maternal MHC H-2d allo-antigens via MMc cell trafficking. This offspring accepted fully allogeneic (H-2d/d ) heart grafts (Andrassy et al., 2003; Dutta and Burlingham, 2011a; Molitor-Dart et al., 2007), showed extended survival of semi-allogeneic skin allografts (Andrassy et al., 2003; Zhang and Miller, 1993), and reduced graft-versus-host disease in bone marrow transplantation (Matsuoka et al., 2006). Further, it was recently suggested that MMc cells might play a role in protection against the development of asthma in humans (Thompson et al., 2013), which could be explained by the migration of allergen-specific MMc T cells that introduce the offspring to this specific allergen, as observed in mice (Fujii and Yamaguchi, 1992; Wan et al., 2002). In this scenario, allo-antigen presentation by MMc results in the induction of long-lasting tolerance. To date, besides solid evidence for the generation of a maternal antigen-specific regulatory T cell compartment in the host (Molitor-Dart et al., 2007) and the suppression of cytotoxic T cell responses (Akiyama et al., 2011; Bonilla et al., 2006), the mechanisms mediating this tolerance are largely unknown.

MMc cells could belong to the antigen presenting cell (APC) compartment. MMc APCs may function in close cooperation with T cells. In addition, it is suggested that the offspring’s APCs could present maternal antigens (Dutta and Burlingham, 2011a; Dutta et al., 2009). In a process referred to as de novo allo-antigen acquisition, professional APCs of the offspring could internalize and re-present maternal antigen at the offspring’s periphery. It has been proposed that this could be one of the mechanisms used to establish tolerance to the MMc allo-antigens that persist until adulthood (Bracamonte-Baran and Burlingham, 2014; Dutta and Burlingham, 2011a; Dutta et al., 2009). Additional hematopoietic lineages are present among the MMc cell pool. Maternal-specific DNA was identified ex vivo and in vitro among CD11b+ myeloid cells (Dutta and Burlingham, 2011a, Dutta et al., 2009; Dutta and Burlingham, 2010) isolated from adult bone marrow and spleen. In the heart, maternal DNA was present among cardiac macrophages scattered across the tissue (Dutta et al., 2009). Also, NK and B cells were found in neonatal and adult lymphoid organs and peripheral blood (Nijagal et al., 2011; Vernochet et al., 2005). Interestingly, in offspring genetically deficient in B cells, MMc cells were a source of IgG-secreting cells (Arvola et al., 2000), providing evidence for the capacity of MMc cells to substitute functionally for immune deficiencies. In line with this, IL-2-deficient offspring acquired IL-2-expressing cells in the thymus and spleen, most suggestively via materno-fetal transmission (Wrenshall et al., 2007). Evidence from human studies indicates that MMc could be replenished during adult life from microchimeric hematopoietic stem cells residing in the offspring’s tissues

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Box 2: Breakdown of tolerance toward selfantigens – a predisposing role in auto-immunity? The engraftment of semi-allogeneic MMc in the offspring’s organs may sensitize the developing offspring’s immune system and may further be involved in the offspring’s predisposition to auto-immune diseases. In human type 1 diabetes, for example, MMc may contribute to the basis of auto-immunity as MMc cells are present at an increased frequency in intact beta cell islets of juvenile patients (Nelson et al., 2007; Vanzyl et al., 2010; Ye et al., 2014b), although their direct participation in the inflammatory response has not been demonstrated. In mice, a model of juvenile type I diabetes showed that MMc T cells with an ovalbumin (OVA)-specific T cell receptor in offspring that expressed OVA in the pancreatic beta cells triggered peri-insulitis in the young adult offspring (Roy et al., 2011). In a similar transgenic approach, highly allo-reactive MMc T cells induced inflammation in the small intestines of 6-8-week-old offspring (Leveque et al., 2014). Antigen-specific MMc T cells may educate the offspring’s immune cells to elicit an immune response against self-antigens through a shift of APCs toward an immunogenic phenotype (Roy et al., 2011) and skew immunity toward auto-reactivity by suppressing the fetal regulatory compartment (Leveque and Khosrotehrani, 2014). A renal auto-immune model suggests that MMc might play an integral part in the pathogenesis of auto-immunity (Lopez-Guisa et al., 2011). Additional studies are necessary to substantiate the long-term role of MMc in the progeny’s regulatory and effector cell compartments, to elucidate the link between human auto-immune diseases and an increased prevalence or frequency of MMc (Artlett et al., 2001; Khosrotehrani et al., 2006; Lambert et al., 2004; Reed et al., 2004; Stevens et al., 2003).

since pregnancy (Hall et al., 1995; Jonsson et al., 2008; Sunku Cuddapah et al., 2010). In mice, maternal DNA was present among lineage− c-Kit+ hematopoietic stem cells in the bone marrow and heart of 6–8-week-old mice (Dutta and Burlingham, 2010; Dutta et al., 2009). Interestingly, a non-hematopoietic mesenchymal stem cell fraction also showed a weak signal for maternal DNA after in vitro expansion of adult mice bone marrow (Dutta and Burlingham, 2010). In rats, adoptively transferred immortalized human mesenchymal stromal cells trafficked through the placenta and homed to a variety of fetal organs, where they persisted up to 3 months postnatally (Chen et al., 2008), providing evidence that mesenchymal stem cells can also reach the fetus through transplacental migration, as discussed above. Thus, a MMc stem cell pool could be the source of maternal cells residing in non-hematopoietic tissue, such as the MMc thymic epithelial cells (Dutta et al., 2009), renal proximal tubular and endothelial cells (Lopez-Guisa et al., 2011), and cardiomyocytes, endothelial, and smooth muscle cells, contained in the lineage− c-Kit− cardiac compartment (Dutta and Burlingham, 2010; Dutta et al., 2009). Overall, not only the location of MMc cells in lymphoid organs, but also their immune phenotype, strongly supports the functional role they play in shaping and

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compensating for the offspring’s immature immune system. Similarly, the detection of MMc hematopoietic cells in non-lymphoid organs implies that they participate in peripheral immune monitoring. Still, their semiallogenicity may provoke an offspring’s immune response in the long term, or, dependent on the MMc cell phenotype, they may themselves be reactive toward the offspring’s antigen, thereby initiating inflammation and predisposing to auto-immunity (Leveque and Khosrotehrani, 2011). The presence of somatic MMc cells expressing tissue-specific antigens not only raises the question of their origin, which may be multipotent stem cells such as the mesenchymal stem cells, but more importantly of their relevance for the developing and long-term physiology of the fully grown organs. 6. What are the factors that modulate the occurrence of MMc? To date, highly variable prevalence and levels of MMc were reported among different experimental set-ups. Differences may be enhanced by the diverse detection approaches, which render the data from separate studies difficult to contrast. Interestingly, a large variability in the frequency of MMc is also observed in different cohorts of human studies (reviewed by Nelson (2012) and in rhesus monkeys (Bakkour et al., 2014)), supporting the idea that a disparity among individuals is inherent to MMc. This disparity may in fact be due to human materno-fetal histocompatibility, which has been shown to intrinsically influence the level of MMc (Berry et al., 2004). In mice, too, the engraftment of MMc cells in lymphoid organs was facilitated to a greater extent in syngeneic offspring compared with allogeneic and outbred offspring (Vernochet et al., 2005). In the kidney, the frequency of MMc cells was higher when they were syngeneic to the fetus than when they were semi-allogeneic (Lopez-Guisa et al., 2011). In comparison to inbred mice, outbred offspring show a rather restricted organ distribution of MMc (Marleau et al., 2003; Piotrowski and Croy, 1996) and a lower frequency of organs containing MMc cells, at least in the postnatal phase (Vernochet et al., 2005). In a different study, allogenicity and H-2 heterozygosity also decreased MMc in the brain and in pooled lymphoid organs (Kaplan and Land, 2005). In immune-deficient offspring, the level of MMc was higher in both lymphoid and non-lymphoid organs compared with immune-competent offspring (Piotrowski and Croy, 1996; Zhou et al., 2000). Thus, the lower the offspring’s immune response against maternal antigens, the higher the level of MMc. This supports the concept that the offspring’s immune system can react against maternal (semi-)allo-antigens and confine their passage, engraftment, and survival. In addition, it reinforces the observation that MMc cells might be capable of substituting for functional immune deficits in the offspring. Interestingly, the occurrence of MMc is also subjected to exogenous modulation. Indeed, intrauterine non-specific tissue injury appears to enhance the transfer of MMc cells or their survival in the fetal organism. In a case of in utero intervention, maternal fetal-specific T cells accumulated in the uterus and its draining lymph nodes (Wegorzewska

et al., 2014) seeming to have sensed the ongoing fetal inflammation. Maternal cell trafficking was increased in in utero sham-injected fetuses compared with unmanipulated offspring (Nijagal et al., 2011), an observation also made in human prenatal surgery (Saadai et al., 2012). MMc was also increased upon pertussis toxin-induced asymptomatic systemic inflammation in pregnant dams (Wienecke et al., 2012). The mechanisms regulating such dynamic changes in MMc are, however, unclear. It is possible that upon inflammation, the increased expression of adhesion molecules in the placenta (reviewed by Solano et al., 2011) could promote the transplacental migration of MMc cells. Nijagal et al. (2011) demonstrated that the phenotype of the MMc cells in fetal circulation differs from that in the maternal blood pool, implying a selective transfer or expansion in the host. Further, MMc cells appeared to selectively proliferate, as demonstrated by their increased quantity and estimated diameter until gd 18.5–19.5 (Marleau et al., 2003) and the scattered localization across the bone marrow, but not in the spleen, thymus, lung, and vertebrae (Piotrowski and Croy, 1996). This, together with the observations of lactational MMc transfer and its multiple phenotypes, supports the existence of mechanisms that control the selective transfer, local expansion, and persistence of MMc cells. Environmental challenges could affect the MMc cell pool that as a messenger mirrors the maternal immune status not only under healthy, but also under adverse conditions. In this scenario, alterations in the MMc population would directly affect the signals it communicates to its fetal recipient. 7. Conclusion: MMc as materno-fetal messenger during pregnancy Growing evidence highlights the clinical relevance of MMc in the context of tolerance and auto-immunity (Boxes 1 and 2). Here, we summarized evidence arising from murine models that could form the basis for understanding the mechanisms underlying these clinical implications. Indeed, extensive kinetic and phenotypical analyses demonstrated that MMc is a physiological and frequent process as MMc cells are present in a variety of offspring tissues commencing at mid-gestation, peaking in peripartum life, and persisting until adulthood. MMc cells can adopt different phenotypes, which may partly result from the local differentiation of stem cells that homed to hematopoietic niches in the offspring. MMc frequency and phenotype can be regulated by intrinsic and extrinsic factors. However, there is little known about the mechanisms that mediate these effects, and, more importantly, the physiological meaning for the host offspring. From the existing evidence, it can be deduced that MMc influences the fetal immune ontogeny, with consequences for the adult offspring’s immune response, e.g., the induction of Tregs. MMc immune cells can protect the fetal organism from foreign challenges and compensate for immature immune function, which strongly suggests a role in the surveillance of the offspring’s developing system. Hence, it is tempting to speculate that MMc might act as a maternofetal messenger executing prenatal immune cross-talk from mother to child, conferring protection during this

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period, while at the same time shaping the offspring’s postnatal physiology. In humans, efforts to elucidate the role of MMc have identified beneficial effects in transplant engraftment and allergy prevention on the one hand. On the other hand, a correlation between MMc and autoimmune diseases suggests a potentially harmful effect on the offspring’s health. We envision that future endeavors in mouse model research will allow the functional characteristics of MMc to be thoroughly investigated in the context of the offspring’s physiology and pathology. Conflicts of interest The authors declare that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. Acknowledgements The authors wish to thank Petra Arck for critically reading the manuscript. The writing of this review and references to our own work was possible because of funding provided by the German Research Foundation (AR232/19), the Werner-Otto Foundation, Cusanuswerk–Bischöfliche Studienförderung, and the Forschungsförderungsfond der Medizinischen Fakultät–Universitätsklinikum HamburgEppendorf. References Akiyama, Y., Caucheteux, S.M., Vernochet, C., Iwamoto, Y., Tanaka, K., Kanellopoulos-Langevin, C., Benichou, G., 2011. Transplantation tolerance to a single noninherited MHC class I maternal alloantigen studied in a TCR-transgenic mouse model. J. Immunol. 186, 1442–1449. Andrassy, J., Kusaka, S., Jankowska-Gan, E., Torrealba, J.R., Haynes, L.D., Marthaler, B.R., Tam, R.C., Illigens, B.M., Anosova, N., Benichou, G., Burlingham, W.J., 2003. Tolerance to noninherited maternal MHC antigens in mice. J. Immunol. 171, 5554–5561. Araki, M., Hirayama, M., Azuma, E., Kumamoto, T., Iwamoto, S., Toyoda, H., Ito, M., Amano, K., Komada, Y., 2010. Prediction of reactivity to noninherited maternal antigen in MHC-mismatched, minor histocompatibility antigen-matched stem cell transplantation in a mouse model. J. Immunol. 185, 7739–7745. Artlett, C.M., Miller, F.W., Rider, L.G., Childhood Myositis Heterogeneity Collaborative Study Group., 2001. Persistent maternally derived peripheral microchimerism is associated with the juvenile idiopathic inflammatory myopathies. Rheumatology (Oxford) 40, 1279–1284. Arvola, M., Gustafsson, E., Svensson, L., Jansson, L., Holmdahl, R., Heyman, B., Okabe, M., Mattsson, R., 2000. Immunoglobulin-secreting cells of maternal origin can be detected in B cell-deficient mice. Biol. Reprod. 63, 1817–1824. Aurrand-Lions, M., Johnson-Leger, C., Lamagna, C., Ozaki, H., Kita, T., Imhof, B.A., 2002. Junctional adhesion molecules and interendothelial junctions. Cells Tissues Organs 172, 152–160. Bakkour, S., Baker, C.A., Tarantal, A.F., Wen, L., Busch, M.P., Lee, T.H., Mccune, J.M., 2014. Analysis of maternal microchimerism in rhesus monkeys (Macaca mulatta) using real-time quantitative PCR amplification of MHC polymorphisms. Chimerism 5, 6–15. Berry, S.M., Hassan, S.S., Russell, E., Kukuruga, D., Land, S., Kaplan, J., 2004. Association of maternal histocompatibility at class II HLA loci with maternal microchimerism in the fetus. Pediatr. Res. 56, 73–78. Bonilla, W.V., Geuking, M.B., Aichele, P., Ludewig, B., Hengartner, H., Zinkernagel, R.M., 2006. Microchimerism maintains deletion of the donor cell-specific CD8+ T cell repertoire. J. Clin. Invest. 116, 156–162. Bracamonte-Baran, W., Burlingham, W., 2014. Non-inherited maternal antigens, pregnancy, and allotolerance. Biomed. J., http://dx.doi.org/ 10.4103/2319-4170.143498.

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