Maternal Microchimerism Histocompatibility and MHC

Influence of Maternal-Fetal Histocompatibility and MHC Zygosity on Maternal Microchimerism

This information is current as Joseph Kaplan and Susan Land of October 1, 2021. J Immunol 2005; 174:7123-7128; ; doi: 10.4049/jimmunol.174.11.7123 http://www.jimmunol.org/content/174/11/7123 Downloaded from References This article cites 31 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/174/11/7123.full#ref-list-1

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Inﬂuence of Maternal-Fetal Histocompatibility and MHC Zygosity on Maternal Microchimerism1

Joseph Kaplan2* and Susan Land†

To investigate the relationship between maternal-fetal histocompatibility and maternal microchimerism, we developed a sensitive quantitative PCR assay for the neomycin resistance gene (neoR), and, in a mouse model system, used neoR as a noninherited maternal allele marker of maternal cells to detect and quantitate maternal microchimerism in tissues of neoR؊/؊ N2 backcross ؉/؊ ؊/؊ progeny of (neoR )F1 females mated with neoR males. Using this approach, we obtained evidence for the presence of chimeric maternal cells in the brain, spleen, and thymus of all weanling and adult mice so tested. The numbers of chimeric maternal cells present in the spleen did not differ significantly from those in the thymus regardless of age or maternal-fetal histocompatibility. At all ages, brain tissue had higher level of maternal microchimerism than lymphoid tissue in mice MHC identical with their mothers, but the levels were similar in mice MHC disparate with their mothers. The levels of chimeric maternal cells in both brain and lymphoid tissue of mice with homozygous syngenicity and maternal allogenicity were similar, and tended Downloaded from to be higher than tissue-specific levels in mice with either combined maternal-fetal allogenicity or heterozygous syngenicity. Thus, MHC homozygous progeny had higher levels of maternal microchimerism than MHC heterozygous progeny. We conclude that normal mice possess small numbers of maternal cells in spleen, thymus, brain, and probably most other tissues, and that maternal- fetal histocompatibility influences the levels of these cells by mechanisms related to MHC zygosity of the progeny. The Journal of Immunology, 2005, 174: 7123–7128. http://www.jimmunol.org/ aternal microchimerism, the presence of very small help fill this gap in knowledge, we have developed a sensitive and fractional concentrations of chimeric maternal cells, accurate quantitative PCR (QPCR)3 assay for the neomycin resis- M occurs in at least half of all humans from fetal life into tance gene (neoR), and have used neoR as a noninherited maternal adulthood (1–4). The factors that govern maternal microchimer- allele (NIMA) in mice to quantitate chimeric maternal cells in Ϫ/Ϫ ϩ/Ϫ ism are poorly understood, but may be of considerable clinical neoR N2 backcross progeny of (neoR )F1 females mated relevance in view of the fact that maternal microchimerism has with neoRϪ/Ϫ males. In the studies described in this work, we have been associated with autoimmunity (5) and allograft tolerance (6), used this approach to examine the relationship between maternal- and may serve as a vehicle for vertical transmission of pathogenic fetal histocompatibility and the prevalence and levels of maternal by guest on October 1, 2021 microbes and allergens (7). microchimerism in lymphoid tissues and brains of weanling and The notion that maternal-fetal histocompatibility plays a role in adult mice. The results indicate that small amounts of NIMA-spe- the regulation of persistent maternal microchimerism derives from cific DNA probably representing maternal cells are present in lym- the well-known importance of donor-host histocompatibility in phoid and nonlymphoid tissues of virtually all neonatal and adult regulating donor cell chimerism in allogeneic bone marrow trans- mice regardless of their pattern of maternal-fetal histocompatibil- plant recipients (8). Much of the evidence for this has come from ity, with a tendency for higher levels in MHC homozygous mice. studies that have taken advantage of the ready availability of in- Consistent with previously reported evidence of the normalcy of bred mouse strains with well-defined MHC genes. There have been maternal cell transfer and long-term engraftment in immunocom- several previously reported investigations of maternal microchi- petent progeny (9), the current findings lend support to the possi- merism in mouse model systems that, taken together, demonstrate bility that maternal cells have a normal physiological role. the frequent occurrence in fetal, neonatal, and adult lymphoid and hemopoietic tissues of chimeric cells transferred in utero and/or Materials and Methods through breastfeeding from allogeneic (9), semiallogeneic (10), Mice and syngeneic (11, 12) mothers. However, mouse models have not Male BALB/cByJ, NZM2410/J, and C57BL/6J (B6) mice age 5 wk were been used to systematically investigate the possible influence of obtained from The Jackson Laboratory. Adult male and female FVIII- maternal-fetal histocompatibility on maternal microchimerism. To knockout mice, homozygous for the neoR gene within the mouse FVIII gene and bred onto the B6 background, were kindly provided by H. Ka- zazian (University of Pennsylvania School of Medicine, Philadelphia, PA). *Carmen and Ann Adams Department of Pediatrics, School of Medicine, and †De- Generation of progeny and tissue harvesting partment of Genetics, Center of Molecular Medicine and Genetics, Wayne State Uni- versity, Detroit, MI 48201 As described below, matings were conducted between various strains of Received for publication October 12, 2004. Accepted for publication March 8, 2005. male and female mice ranging in age from 2 to 5 mo. Each of the female mating partners in this study had produced one to two previous litters The costs of publication of this article were defrayed in part by the payment of page derived from matings with the same strains of males. All progeny were charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. weaned at age 22 days. At 0–45 days of age, all progeny of a single litter were sacrificed, and specimens of spleen, thymus, brain, and bone marrow 1 This research was supported by a grant from the Children’s Research Center of were individually quick frozen in liquid nitrogen for subsequent individual Michigan. 2 Address correspondence and reprint requests to Dr. Joseph Kaplan at the current address: Children’s Hospital of Michigan, 3901 Beaubien Boulevard, Detroit, MI 3 Abbreviations used in this paper: QPCR, quantitative PCR; NIMA, noninherited 48201. E-mail address: [email protected] maternal allele.

Ϫ Ϫ NIMA testing. The 0- to 45-day age range was chosen to test for possible neoR / wild-type males. As a first step, we sought to develop a quantitative and tissue-specific differences between short-term neonatal sensitive and accurate QPCR method for detection of the neoR and long-term adult persistence of maternal microchimerism because any gene. We first generated obligatory neoRϩ/Ϫ heterozygotes by such differences might reflect age-related host immune effects on the sur- ϩ/ϩ vival of chimeric maternal cells in offspring, mating neoR homozygous B6 FVIII-knockout females with wild-type B6 males. A known amount of genomic DNA isolated DNA isolation from tissue derived from a neoRϩ/Ϫ mouse was then serially di- Samples of DNA from frozen aliquots of splenic, thymic, brain, and bone luted in a fixed known amount of genomic DNA from a wild-type marrow tissues were individually prepared from each mouse using neoRϪ/Ϫ mouse, and each dilution was used as genomic DNA in QIAamp DNA Extraction kits for PCR (Qiagen), according to the manu- QPCR assays for the neoR gene. To standardize the assay for facturer’s directions. Each organ from each mouse was analyzed separately. genomic DNA, a TaqMan assay for rodent GAPDH available from Applied Biosystems was run in parallel with the neoR TaqMan NeoR genotyping assays on each of the DNA samples. The results showed a tight fit In some experiments, DNA samples were genotyped for neoR sequences to linearity (Fig. 1), sensitivity to the level of detection of one neoR by conventional PCR using neoR forward primer, 5Ј-CTA TGT CCT GAT genome equivalent, and neoR specificity because PCR product was AGC GGT CC-3Ј and neoR reverse primer, 5Ј-CGG CTG CAT ACG CTT only detected with DNA from neoRϩ mice; none was detected Ј GAT CC-3 . With these primers, amplification of DNA from neoR-positive after 40 cycles of real-time PCR using DNA from wild-type neoRϪ progeny yields a 250-bp product. B6 mice. Based on these results, subsequent experiments used H-2 genotyping QPCR assays instead of standard PCR assays to determine the neoR genotype of backcross progeny of all neoRϩ/Ϫ ϫ neoRϪ/Ϫ

Mouse DNA samples were genotyped for H-2, as previously described Downloaded from (13), using PCR amplification of highly polymorphic microsatellites in the matings. second intron of the H-2Eb gene. Positive controls for H-2b, H-2d, and Ϫ Ϫ H-2z consisted of DNA isolated from spleen cells obtained from B6, Generation of NIMA informative neoR / progeny differing in BALB/c, and NZM mice. maternal-fetal histocompatibility QPCR analysis Four different backcross mating schemes were used to generate Ϫ/Ϫ ϩ/Ϫ QPCR analysis was performed using the TaqMan PCR Core Reagent kit neoR progeny of neoR mothers with different maternal- http://www.jimmunol.org/ (Applied Biosystems). Reactions for neoR-genomic DNA quantification fetal histocompatibility relationships across the entire H-2 com- were performed in 30 ␮l with 0.5 ␮g of DNA; 3 ␮lof10ϫ TaqMan buffer plex (Table I). The terms used in this work to describe these ma- ␮ A (500 mM KCl, 100 mM Tris-HCl, pH 8.3); 5 mM MgCl2; 200 M each ternal-fetal histocompatibility relationships, homozygous of dATP, dCTP, and dGTP; 400 ␮M dUTP; 0.3 U of uracil-N-glucosidase; 0.75 U of AmpliTaq Gold DNA polymerase; 50 nM probe; and 100 nM syngenicity, heterozygous syngenicity, fetal semiallogenicity, ma- sense and antisense primers. NeoR gene nucleotide sequence data obtained ternal semiallogenicity, and combined maternal and fetal semial- from the GenBank database (accession U52109) and Primer Express soft- logenicity, are based on those described by Hoff et al. (14). All ware (Applied Biosystems) were used to design the following neoR-spe- members of a given litter of N2 progeny were killed on the same cific oligonucleotide probe and forward and reverse PCR primers specific Ј day, and samples of their spleen, thymus, and brain were snap for an invariant section of the neoR transcript: neoR sense primer, 5 -TGA Ϫ GCC TGG CGA ACA GTT C-3Ј; neoR antisense primer, 5Ј-CCG GTC frozen and stored at 80°C. Aliquots of genomic DNA isolated by guest on October 1, 2021 TTG TCG ATC AGG AT-3Ј; and neoR probe, 5Ј-6FAM-ATG CTC TTC from frozen tissues were used in H-2-specific PCR to determine GTC CAG AT-MGBFQ-3Ј. All were purchased from Applied Biosystems. the H-2 genotypes of the tissue donors, and in neoR-specific QPCR The QPCR program used an initial temperature of 50°C for 2 min and then assays to determine their neoR genotypes, and to detect and quan- 95°C for 10 min, followed by 40 amplification cycles run for 15 s at 95°C neoR and 1 min at 60°C. The amplifications were performed on an ABI Prism titate NIMA-specific DNA in tissues of mice found to be 7700 sequence detector equipped with a 96-well thermal cycler. Data were negative. collected and analyzed with Sequence Detector v1.6.3 software (Applied Biosystems). As a reference control, a TaqMan QPCR assay for murine Effects of tissue source and donor age on prevalence and levels GAPDH was run in parallel on each DNA sample using probes and primers of maternal microchimerism available from Applied Biosciences. Quantitation of the fractional amount of neoR genomic DNA present in each DNA sample was calculated using We detected at least some NIMA-specific DNA in the brain, the relative standard curve method. The amount of neoR present in DNA spleen, and thymus of all weanling and adult mice so tested. The ϩ Ϫ from a single neoR / cell was considered one genome equivalent of neoR, fractional levels of NIMA-specific DNA in each of these tissues and the amount of GAPD in one diploid cell was considered one genome equivalent of GAPDH. The fractional concentration of neoRϩ/Ϫ cells rep- resented in each sample of DNA was calculated as a ratio to the GAPD genome equivalents detected in the same DNA sample. All of the assays were done at two different concentrations in duplicate with probes labeled with FAM and TAMRA. Statistical analysis Differences in the means of the log-transformed fractional levels of NIMA- specific DNA were compared by ANOVA or by the Mann-Whitney U test. Fisher’s protected least significant difference test was used for multiple comparisons with significance set at p Ͻ 0.05. All statistical analyses were conducted using the StatView 4.5 software program for Macintosh computers. Results Development of a 5Ј nuclease (TaqMan) QPCR assay for the FIGURE 1. Standard curve for QPCR assay for the neoR gene. The neoR gene graph plots Ct, the threshold values of the PCR cycle number first detecting signal for amplified genomic DNA in the QPCR, against log (concentration Our overall strategy in these experiments was to use the neoR gene of genomic DNA) of serially diluted samples of DNA from a neoR-positive as a NIMA to quantitate the levels of chimeric maternal cells in mouse. The plot shows tight linearity and sensitivity of detection to one Ϫ Ϫ ϩ Ϫ neoR / N2 backcross progeny of neoR / females mated with genome equivalent. The Journal of Immunology 7125

Table I. Mating schemes

H-2 Genotype(s) NeoRϩ/Ϫ Mother NeoRϪ/Ϫ Father Progeny Maternal-Fetal Histocompatibility

ϩ/ϩ ϫ (B6neoR B6)F1 (H-2b/b) B6 (H-2b/b) H-2b/b Homozygous syngenicity ϩ/ϩ ϫ (B6neoR BALB/c)F1 (H-2b/d) BALB/c (H-2d/d) H-2b/d Heterozygous syngenicity H-2d/d Maternal semiallogenicity ϩ/ϩ ϫ (B6neoR BALB/c)F1 (H-2b/d) B6 (H-2b/b) H-2b/d Heterozygous syngenicity H-2b/b Maternal semiallogenicity ϩ/ϩ ϫ (B6neoR NZM)F1 (H-2b/z) CByB6F1 (H-2b/d) H-2b/z Heterozygous syngenicity H-2b/b Maternal semiallogenicity H-2b/b Maternal/fetal semiallogenicity H-2z/d Maternal/fetal semiallogenicity showed no relationship to age, nor were there any differences be- erozygous syngenicity ( p ϭ 0.004). As pointed out above, across tween the levels detected in spleen and thymus. Therefore, we all mice tested, brain tissue had significantly higher fractional lev- pooled data from mice of all ages, and from spleen and thymus, els of NIMA-specific DNA than lymphoid tissue. However, when and compared data from this pooled lymphoid tissue with that analyzed for the effect of maternal-fetal histocompatibility, this obtained from brain. As shown by regression analysis (Fig. 2), the difference was only significant in the case of mice with histocom- levels of NIMA-specific DNA in brain correlated with those in patible mothers, i.e., those with maternal-fetal homozygous syn- Downloaded from lymphoid tissue. genicity ( p Ͻ 0.0001) or heterozygous syngenicity ( p ϭ 0.0013), and not in those with maternal semiallogenicity ( p ϭ 0.94) or Effects of maternal-fetal histocompatibility on levels of NIMA- maternal-fetal semiallogenicity ( p ϭ 0.69). specific DNA in brain and lymphoid tissue The results presented in Table II and Figs. 3 and 4 compare the fractional levels of NIMA-specific DNA in brain and lymphoid Discussion http://www.jimmunol.org/ tissue from mice generated by pregnancies with different types of In the studies described in this work, we developed a highly sen- maternal-fetal histocompatibility. The data for each maternal-fetal sitive QPCR assay for quantitation of the neoR gene, and used the histocompatibility type represent pooled data from the different neoR gene as a NIMA in mouse model experiments designed to mating schemes generating each of these types because we de- test for the effects of maternal-fetal histocompatibility on the pres- tected no significant within-type differences. The fractional levels ence and levels of maternal microchimerism. Our strategy was to of NIMA-specific DNA in both brain and lymphoid tissue of mice use NIMA-specific DNA as a marker of chimeric maternal cells to with homozygous syngenicity and maternal semiallogenicity were assess the prevalence and fractional amount of maternal microchi- similar, and tended to be higher than in mice with either combined merism in the brain and lymphoid tissue of informative homozy- by guest on October 1, 2021 maternal-fetal semiallogenicity or heterozygous syngenicity. By gous neoRϪ/Ϫ backcross progeny of heterozygous neoRϩ/Ϫ female ANOVA, the mean fractional level of NIMA-specific DNA in the mice mated with male wild-type neoRϪ/Ϫ mice. The mating part- brain was significantly higher in mice with homozygous syngenic- ners were chosen to be homozygous or heterozygous at the MHC ity than in mice with either combined maternal-and-fetal semial- locus for either shared or disparate alleles to create progeny with logenicity ( p ϭ 0.016) or heterozygous syngenicity ( p ϭ 0.05). In lymphoid tissue, as in the brain, the mean fractional level of four possible patterns of maternal-fetal histocompatibility. Using NIMA-specific DNA in mice with maternal-fetal homozygous this approach, we detected NIMA-specific DNA in the brain and syngenicity was significantly higher than in mice with maternal- lymphoid tissue of all adult mice so tested regardless of the pres- fetal heterozygous syngenicity ( p ϭ 0.02), and tended to be higher ence or absence of maternal-fetal histocompatibility. However, than in mice with combined maternal-and-fetal semiallogenicity comparison of the fractional levels of NIMA-specific DNA in mice ( p ϭ 0.08). Lymphoid tissue levels were also significantly higher derived from pregnancies with different types of maternal-fetal his- in mice with maternal semiallogenicity than in mice with either tocompatibility revealed a consistent pattern: regardless of their combined maternal-and-fetal semiallogenicity ( p ϭ 0.02) or het- age at testing, the levels in mice derived from pregnancies with maternal-fetal homozygous syngenicity and maternal semiallogenicity were similar, and tended to be higher than in mice derived from pregnancies with either combined maternal-fetal semiallogenicity or heterozygous syngenicity. It seems likely that most, if not all, of the NIMA-specific DNA detected in these studies derives from intact chimeric cells, and thereby provides an accurate reflection of the presence and level of maternal microchimerism. However, in the absence of direct evidence for chimeric cells using such techniques as in situ immuno- histochemistry and immunofluorescence, it remains possible that at least some of the NIMA-specific DNA represents cell-free DNA released from dead cells either in situ or in the circulation. This is a particularly important consideration with regard to the data ob- FIGURE 2. Direct correlation between levels of maternal microchimer- tained in this study with brain tissue, because, while direct evi- ism in brain and lymphoid tissue. Data represent Ln (fractional levels of dence for chimeric maternal cells in lymphoid tissues of adult NIMA-specific DNA ϫ 10E-5) detected by real-time PCR in brain and progeny has been previously reported (10), the current findings are lymphoid tissue (spleen and/or thymus) in 16 mice. the first evidence supporting the presence of chimeric maternal 7126 MATERNAL-FETAL H-2 COMPATIBILITY AND MATERNAL MICROCHIMERISM

Table II. Fractional levels of maternal microchimerism in brain and lymphoid tissue of progeny with different types of maternal-fetal histocompatibility

Maternal-Fetal Histocompatibility Brain Lymphoid Tissuea

Homozygous syngeneic (Homo S) 5.31 Ϯ 0.47b,c (n ϭ 15) 3.30 Ϯ 0.21d (n ϭ 49) Heterozygous syngeneic (Hetero S) 3.93 Ϯ 0.43 (n ϭ 13) 2.51 Ϯ 0.19 (n ϭ 34) Maternal semiallogeneic (MA) 3.79 Ϯ 0.88e (n ϭ 6) 3.73 Ϯ 0.34f,g (n ϭ 23) Maternal/fetal semiallogeneic (MFA) 2.92 Ϯ 0.94h (n ϭ 5) 2.50 Ϯ 0.52i (n ϭ 15)

a Pooled data from spleen and thymus. b Ln fraction of NIMA-specific DNA ϫ 10Ϫ5 (mean Ϯ SE). c p ϭ 0.05 Homo S vs Hetero S (brain). d p ϭ 0.02 Homo S vs Hetero S (lymphoid tissue). e p ϭ 0.09 Homo S vs MA (brain). f p ϭ 0.001 MA vs MFA (lymphoid tissue). g p ϭ 0.003 MA vs Hetero S (lymphoid tissue). h p ϭ 0.016 Homo S vs MFA (brain). i p ϭ 0.08 Homo S vs MFA (lymphoid tissue). cells in the brain. The existence of the blood barrier to large mol- comes from the observation that brain tissue had significantly ecules and the extremely short t1/2 of foreign DNA in the circu- higher fractional levels of NIMA-specific DNA than lymphoid tis- lation (15) makes it unlikely that the NIMA-specific DNA detected sue in mice with histocompatible mothers, but not in mice with Downloaded from in this study in brain represents circulating DNA released into the histoincompatible mothers. Consistent with the findings of Mar- circulation by dead cells outside the brain. Moreover, even if the leau et al. (9) that the prevalence and level of maternal microchi- NIMA-specific DNA derives in part from DNA released in situ by merism in extramedullary tissues are lower and more transient than chimeric cells, the data still support the presence of such cells in in bone marrow in immunocompetent offspring of MHC disparate the brain. The only previously reported data bearing on this ques- mothers, but not in immunodeficient or histocompatible offspring, tion is that of Piotrowski and Croy (11), who, using histochemical this finding also suggests that NIMA-specific T cell regulation of http://www.jimmunol.org/ methods, failed to detect maternal cells in sections of fetal mouse chimeric maternal cells may play a more important regulatory role brain tissue obtained from gestation day 8 to term. The discrepancy in some tissues than others. between the results of that previous study and the data obtained in Although classic T and B cell alloreactivity to noninherited ma- this study could be due to a lower sensitivity of NIMA detection by ternal MHC Ags clearly plays a role in regulating chimeric ma- histochemistry compared with PCR. ternal cells, the overall pattern of similarities and differences in the The current data do not permit estimation of the extent to which levels of maternal microchimerism detected in this study in mice the NIMA-specific DNA detected in this study represents maternal derived from pregnancies with different types of maternal-fetal his- cells derived from prenatal transplacental passage or maternal cells tocompatibility cannot be readily explained by any single mecha- by guest on October 1, 2021 derived from postnatal lactational transfer. However, previously nism involving unidirectional or bidirectional maternal-fetal T or reported evidence that prenatally acquired maternal cells have a B cell alloreactions. The expected T and B cell tolerance to self greater capacity for long-term postnatal persistence than those ac- MHC Ags in adult mice derived from pregnancies with homozy- quired postnatally (10, 16) taken together with the observation that gous syngenicity and the expected T and B cell alloreactivity to chimeric maternal cells first appear and are most prevalent in the noninherited maternal MHC Ags of adult mice derived from preg- bone marrow (17) favors the notion that the NIMA-specific DNA nancies with combined maternal and fetal semiallogenicity could detected in this study in adult lymphoid tissue and brain represents readily account for the higher levels of maternal microchimerism maternal cells that had originally seeded and successfully en- detected in the former than in the latter, but none of these mech- grafted the fetal bone marrow in utero. anisms explains why progeny of homozygous syngeneic pregnan- Our detection of NIMA-specific DNA in progeny with maternal cies have higher levels of persistent maternal microchimerism than semiallogenicity fits with findings previously reported by Zhang progeny of heterozygous syngeneic pregnancies in both brain and and Miller (10), who, using immunofluorescence assays, detected lymphoid tissue. Homozygous syngeneic progeny and heterozy- small numbers of maternal cells expressing the noninherited ma- gous syngeneic progeny should both be tolerant to maternal cells ternal H-2d allele in lymph nodes of many, but not all, 6-wk-old because such cells presumably express exactly the same comple- b b/d H-2 backcross offspring of H-2 B6D2F1 females mated with ment of MHC Ags as cells from the progeny themselves. Differ- H-2b/b B6 males. The greater prevalence of maternal microchimer- ences in the presence or absence of T or B cell self-tolerance and ism suggested by our results is probably related to a higher sen- alloreactivity also fail to explain why the level of maternal micro- sitivity of detection of NIMA by PCR than by direct chimerism is higher in mice with maternal allogenicity than in immunofluorescence. mice with either heterozygous syngenicity or combined maternal- The occurrence of maternal microchimerism in mice in the face fetal allogenicity. of maternal-fetal histoincompatibility is also entirely consistent Further analysis of the data suggests that zygosity at the MHC with similar findings in humans (18–20). In both species, this can locus is the one histocompatibility characteristic that distinguished be explained, in part, by the fact that partial tolerance to nonin- progeny with relatively high vs those with relatively low levels of herited maternal alloantigens is a normal occurrence. Thus, both maternal microchimerism. Thus, as shown in Fig. 4, compared human and mouse studies indicate that in utero and neonatal ex- with progeny with MHC heterozygosity (i.e., those with H-2b/d, posure to NIMA-expressing cells frequently induces long-lasting H-2b/z,orH-2z/d genotypes), mice with MHC homozygosity (i.e., NIMA-specific transplant tolerance in vivo (6, 10, 21, 22) and those with H-2b/b or H-2d/d genotypes) had significantly higher reductions in the frequency and functional activity of NIMA-spe- levels of maternal microchimerism in both brain ( p ϭ 0.05) and cific T cells (22, 23). In the current study, support for a role for lymphoid tissue ( p ϭ 0.004). It can be argued that the zygosity NIMA-specific T cells in regulating maternal microchimerism effects on maternal microchimerism observed in this study are due The Journal of Immunology 7127

FIGURE 4. Levels of chimeric maternal cells are higher in progeny with MHC homozygosity than in progeny with MHC heterozygosity in both brain (p ϭ 0.05) and lymphoid tissue (p ϭ 0.004). Blocks contain interquartile range and means. Bars indicate SEM. Dots are outliers. FIGURE 3. Relationship between type of maternal-fetal histocompatibility and levels of maternal cells in brain and lymphoid tissue. MA, Ma- ternal allogenicity; MFA, combined maternal and fetal allogenicity; hetero S, heterozygous syngenicity; homo S, homozygous syngenicity. Blocks

demonstration of the ubiquity and widespread tissue distribution of Downloaded from contain interquartile range and means. Bars indicate SEM. Dots are chimeric maternal cells in both weanling and adult mice, this fits outliers. with the possibility that small numbers of chimeric maternal cells may have the beneficial effect throughout an offspring’s life of participating in tissue repair and regeneration. to zygosity effects of non-MHC genes rather than zygosity effects If maternal microchimerism occurs throughout life, and has the of MHC genes. However, this seems unlikely because identical potential for exerting both beneficial tissue reparative effects and http://www.jimmunol.org/ MHC zygosity effects were observed in comparisons of several deleterious autoimmune effects, what might determine the balance different types of MHC homozygous and heterozygous progeny between the two? That scleroderma in women is associated with even though the relative maternal and fetal zygosity of most of increased levels of fetal microchimerism, but not increased prev- their non-MHC-linked genes should not necessarily correspond to alence of fetal microchimerism (30), suggests that autoimmunity is the relative maternal and fetal zygosity of their MHC genes. For- associated with relatively high levels of normally occurring mi- mal confirmation of a role for MHC zygosity in regulating mater- crochimerism. If so, the relatively lower levels of maternal micro- nal microchimerism could come from studies similar to those de- chimerism observed in this study in association with MHC het- scribed in this work comparing the levels of chimeric maternal erozygosity may account in part for the recently described cells in MHC congenic mice with different genetic backgrounds. protective effect of MHC heterozygosity on autoimmunity (31). In by guest on October 1, 2021 The apparent importance of MHC zygosity in regulating in vivo light of this possibility, and the association of autoimmunity with levels of persistent maternal microchimerism highlights the likely both maternal and fetal microchimerism, it will be of interest to importance of host NK cells in this phenomenon because, in con- determine whether MHC zygosity affects fetal microchimerism in trast to T and B cell specificity, NK cell specificity is determined the same manner as it affects maternal microchimerism. in many cases by the relative MHC zygosity of NK cells and their targets (24, 25). For example, transplanted fully allogeneic cells Disclosures are rapidly removed by mouse and rat NK cells, whereas semial- The authors have no financial conflict of interest. logeneic cells usually are not (26–28). Based on such findings, Zhang and Miller (10) previously pointed out that this pattern of References NK specificity favors survival of maternal cells in offspring be- 1. Socie, G., E. Gluckman, E. Carosella, Y. Brossard, C. Lafon, and O. Brison. cause such cells are normally semiallogeneic rather than fully al- 1994. Search for maternal cells in human umbilical cord blood by polymerase logeneic, and presented evidence directly supporting that chain reaction amplification of two minisatellite sequences. Blood 83: 340–344. 2. Scaradavou, A., C. Carrier, N. Mollen, C. Stevens, and P. Rubinstein. 1996. hypothesis. Detection of maternal DNA in placental/umbilical cord blood by locus-specific All of the mice tested in this study resulted from pregnancies amplification of the noninherited maternal HLA gene. Blood 88: 1494–1500. yielding NeoR-positive as well as NeoR-negative progeny, and de- 3. Petit, T., M. Dommergues, G. Socie, Y. Dumez, E. Gluckman, and O. Brison. 1997. 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