Meeting Report
Chimerism 4:4, 132–135; October/November/December 2013; © 2013 Landes Bioscience
Meeting report of the First Symposium on Chimerism Astrid GS van Halteren1,*, Peter Sedlmayr2, Thomas Kroneis2, William J Burlingham3, and J Lee Nelson4 1 Department of Pediatrics; Willem Alexander Children’s Hospital; Leiden University Medical Center; Leiden, the Netherlands; Institute of Cell Biology, Histology and Embryology; University of Graz; Graz, Austria; 3Department of Surgery; Division of Transplantation; University of Wisconsin School of Medicine and Public Health; Madison, WI USA; 4Fred Hutchinson Cancer Research Center; University of Washington; Seattle, WA USA
Keywords: (micro)chimerism, FISH, HLA, PCR, autoimmune disease, cancer, cell trafficking, pregnancy, tissue repair, transplantation
Introduction The incidence and potentially different biological impact of naturally acquired maternal and fetal cells were enthusiastically and extensively discussed when basic scientists and clinicians met at the Medical University of Graz in Austria for an International Symposium on Chimerism (Fig. 1). The two day program featured presentations by speakers from 4 different continents, intermingled with short presentations focusing on technical aspects of chimerism analysis by representatives from the companies who sponsored the meeting. Details on the program and speakers list are available at the meeting’s website (http:// chimerism.medunigraz.at/cms/website.php). As also exemplified by the various different types of studies published in the recently released journal Chimerism, this field is slowly but steadily expanding as new research teams working outside and within the transplantation field have become interested in the biologically intriguing phenomena of chimerism and microchimerism (Mc). The highlights of the meeting are summarized in this meeting report.
Technical Aspects of Chimerism Analysis Since the 1979 paper by Herzenberg and colleagues reporting enrichment of cells from pregnant women presumed to be fetal using flow-sorting techniques,1 many different studies have been published describing fetal or maternal cells in diverse hematopoietic cell types or tissues and organs. During the past decade, major technical improvements regarding the detection of these generally rare cells have been made. This has resulted in a shift from the more traditional XY fluorescent in situ hybridization (FISH) staining technique applied to tissue sections to more refined technologies such as real-time PCR for the detection of male-specific DNA sequences in genomic DNA extracted from peripheral blood cells or processed tissues. Realtime PCR reactions for mismatched HLA alleles as well as for other genetic markers (insertion/deletion) have been developed *Correspondence to: Astrid GS van Halteren; Email: [email protected] Submitted: 11/11/2013; Accepted: 11/12/2013 http://dx.doi.org/10.4161/chim.27168
in the laboratories of meeting participants Dr Nelson (Fred Hutchinson Cancer Research Center, Seattle, USA), Dr Lambert (INSERM UMRs 1097, Marseille, France) and Dr van Halteren (Leiden University Medical Center, Leiden, the Netherlands). Unfortunately, many researchers are faced by the problem that this approach requires the availability of DNA from mother or offspring in order to find informative genetic markers. Of note, the newly developed QuantiChimera platform established at the laboratory of Dr Lambert can be contacted for technical support regarding state-of-the art PCR technology for Mc detection. Particularly relevant for investigators who are restricted to using FISH techniques for the identification of rare maternal or fetal chimeric cells is the collective observation that this technique does not always give consistent staining results. This may be explained by the fact that archived tissue samples are often subjected to different fixation protocols. As discussed by Dr Sedlmayr and Dr Kroneis (Medical University, Graz, Austria), FISH by itself is a less than optimal technique for the analysis of rare microchimeric cells given the frequency of false positive cells. This issue can in part be addressed by using two reverse sets of differently labeled X and Y probes. Also biochemical markers such as unique cell surface molecules or markers that can be used to distinguish fetal cells from adult cells are not perfectly reliable in the setting of analysis of extremely rare cells. Unambiguous identification of individual microchimeric cells can be done by combining either FISH analysis or analysis of biochemical markers with DNA genotyping of candidate cells isolated by laser microdissection.2 The introduction of a whole genome amplification step subsequently even allows combining DNA genotyping of single microchimeric cells with sequencing or comparative genome hybridization.3 This exciting new approach is expected to further boost the chimerism field.
Why Our Natural Immigrants Matter The main route of acquiring chimeric cells from mother and/or fetus is placental exchange of cells during pregnancy. As presented by Dr Hahn (University Hospital Basel, Basel, Switzerland) fetal cell trafficking to the mother starts already very early in pregnancy and is significantly increased in women with placental defects including preeclampsia.4 Using HLA-specific PCR, several groups have now collected evidence strongly suggesting
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2
Figure 1.The Symposium on Chimerism. Photo courtesy of Harry Schiffer, Copyright Graz Tourism.
that male cells can also be acquired from an older brother (transmaternal cell trafficking)5 or even from a twin brother who died during embryonic development (“vanished” twin). As discussed by Dr Nelson, particularly adult women may bear chimeric blood cells from multiple sources6. Intriguingly, maternal cells acquired during fetal/neonatal life seem to be replaced by fetal-derived cells when females enter the reproductive phase. Alternatively, pregnancy in later life may induce redistribution of maternal cells in tissues. As outlined in Figure 2, these naturally acquired fetal or maternal cells may affect normal biological processes such as tissue repair and reproduction, but also seem to play a role in an array of pathological responses occurring later in life; the latter include disease features associated with autoimmunity, infection, transplantation and cancer. These topics were addressed at the meeting. Tissue repair Mechanistic studies on chimeric cell trafficking and function in experimental animal models are now feasible due to the generation of green fluorescent protein (GFP) reporter mice, which enable researchers to visualize and study the biological impact of fetal-maternal and maternal-fetal cell trafficking in vivo. Mouse studies on fetal cell contribution to tissue repair were nicely presented by Dr Khosrotehrani (University of Queensland, Brisbane, Australia) and Dr Chaudhry (Mount Sinai School of Medicine, New York, USA). Dr Khosrotehrani’s research focuses on fetal cell trafficking to experimentally induced inflammation to tissues or organs.7 Using bioluminescence imaging, data from his group showed that GFP-tagged fetal mesenchymal cells acquired by female mice through pregnancy are recruited to experimentally injured skin and kidney tissue where they respectively produce collagen and induce angiogenesis of small blood vessels comprised of maternal and fetal endothelial cells. Dr Chaudhry’s group is studying fetal cell migration and in situ differentiation of these cells to understand the high recovery rates of peripartum cardiomyopathy, an inflammatory condition which may occur during the last stage of pregnancy up to 6 mo after delivery.8 Using GFP-reporter mice, she elegantly showed that cells originating from the fetus home to the hearts of female mice with experimentally induced myocardial injury.
Meeting Report
These cells express various types of proteins which are typically expressed by cardiomyocytes. Furthermore, GFP+ cells isolated from maternal hearts form vascular tubes and even form beating cardiomyocytes when cultured on feeder layers. Remarkably, a significant percentage of fetal cells present in maternal hearts express transcription factors such as Cdx2 which is typically expressed by placental stem cells (trophoblast stem cells). Whether placenta-derived Cdx2 expressing cells can be used in cardiovascular regenerative therapy is currently under investigation. Microchimerism in autoimmune disease Mc, identified in peripheral blood and tissues, has been strongly implicated in systemic sclerosis.6 As presented by Dr Lambert, genome-wide association studies have revealed that HLA-DRB1*1104 is associated with the highest risk of scleroderma in Caucasian populations. One of the still unsolved issues is the observation that women who have given birth to an HLA-compatible child are at an increased risk of developing systemic sclerosis. This observation opened the discussion whether HLA compatibility in several generations of one family facilitates the establishment or expansion of Mc. A large study is needed to address this issue. In addition, it has been suggested that Mc may have a “mini-gene transfer” like effect, conferring risk or protection from a disease depending on the particular HLA allele carried by the Mc,9 as discussed by Dr Lambert and colleagues. Another unsolved question is whether or not the same microchimeric cells exert different biological effects. Microchimeric cells, either from fetal or maternal origin, can be seen as circulating or tissue-residing targets for host-derived pathological T cells resulting in autoimmune inflammation. This possibility was discussed by D. Gillespie (University of Bristol, Bristol, UK) who presented data demonstrating that maternal Mc in peripheral blood, as measured by real-time PCR specific for non-inherited maternal HLA sequences, is increased in children with type 1 diabetes. Combining XY FISH and immunostaining on pancreatic tissue from individuals with type 1 diabetes and healthy controls, she showed that tissue-residing maternal cells express tissue-specific markers including insulin (marking β cells) or glucagon (marking α cells) but CD45+ve cells in the insulitic infiltrate were not maternal.10 These results suggest that pancreas-residing, partially HLA mismatched, chimeric cells are not the effectors of the autoimmune destruction process, but rather perhaps are involved in the initiation of autoimmunity by triggering maternal alloreactive T cells. Alternatively, chimeric cells may facilitate the repair of damaged cells in the islets of Langerhans. These maternal microchimeric cells have been observed clustered in small sections of the islets. Whether this pattern somehow prevents destruction of all insulin-producing β cells remains speculative. Another interesting discussion point as raised by Dr Hahn is the possibility that the maternal Mc carry a virus which may trigger the onset of insulitis. Mc can somehow decrease the risk of autoimmune disease. This dual action is best exemplified in the setting of Rheumatoid Arthritis (RA) as discussed by Drs Lambert and Nelson. Women who lack RA susceptibility HLA alleles9 (encoding QKRAA
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Meeting Report
Figure 2. The Jing Jang of naturally acquired microchimerism, some examples of fetal and maternal microchimerism that were discussed. Fetal or maternal microchimeric cells may positively affect normal biological responses and/or protect the person who harbors the microchimerism from pathological challenges. On the other hand, microchimerism may also play a negative role by directly or indirectly contributing to immunopathology.
(Leiden University Medical Center, Leiden, the Netherlands), all presented data favoring the concept that microchimeric cells can have a profound effect on the host’s alloreactive T cell repertoire. Dr Burlingham proposed a new term for alloantigens presented by microchimeric cells: Naturally Acquired Chimerism-Derived Occult Antigens, NACHOs. Using carefully selected mouse breeding models, work from his group has clearly demonstrated that NACHOs-expressing maternal microchimeric cells can be found in peripheral blood, bone marrow (c-kit+ progenitor cells) and in various organs such as the heart. The percentage of organs expressing maternal Mc correlates with the degree of immune regulation to non-inherited maternal antigens (NIMA) expressed by these chimeric cells. Surprisingly, they found that maternal Mc was difficult to detect in lymphoid organs such as spleen, lymph nodes, and thymus. However spleen, lymph node and blood contained a relatively high proportion of host MHC class II expressing cells that had acquired maternal antigens. This NIMA-specific antigen acquisition may provide the missing link between Mc and tolerance to a subsequent transplant. T cells that mediate tolerance to NIMA+ allografts can be detected by transvivo delayed type hypersensitivity (tvDTH) testing. This assay works well for analyzing the prevalence and function of both murine14 and human15 T regulator cells (Treg). An instruction video on the principles of this assay can be seen at the following website: http://www.jove.com/video/4454/trans-vivo-delayedtype-hypersensitivity-assay-for-antigen-specific. As presented by Dr Burlingham, the pre-transplant existence of NIMA tolerance, as indicated by the presence of tissueresident maternal Mc acquired during fetal development and
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or QRRAA sequences) can acquire Mc that encodes RA susceptibility alleles and this type of Mc is thought to increase RA risk. Other studies suggest, on the other hand, that if Mc encoding a HLA sequence which is protective against RA (i.e., DERAA encoding HLA alleles) is acquired, the presence of such cells may decrease the risk of developing RA. In both cases, microchimeric cells could be acquired from an individual’s mother or, in women, from the woman’s own prior pregnancies. Chimeric cells acquired from the mother during fetal life occur at a phase when the immune system is still developing. In contrast, fetal cells exchanged during pregnancy are encountered by a fully matured maternal immune system. Whether or not these two sources of Mc indeed differ remains a question of interest for further studies. As discussed by Dr Fugazzola (University of Milan, Milan, Italy), fetal Mc is also detected at higher frequency in autoimmune thyroid disease (Graves disease and Hashimoto thyroiditis) compared with non-autoimmune disease-affected thyroid tissues and post-mortem analyzed normal thyroid glands.11 The limited data available on fetal Mc in autoimmune thyroid diseases at the peripheral blood level seem to indicate its pathogenic role.12 In contrast to earlier studies on this topic, data from the Milan group showed a prevalence of circulating fetal Mc that was actually lower in women with Graves disease as compared with healthy controls. This observation suggests there may be more complexity to a possible role of fetal Mc in Graves’ disease. Dr Stevens (University of Washington, Seattle, USA) presented data on the role of Mc in children with systemic lupus erythematosus (SLE). Her group is studying maternal Mc13 as well as Mc originating from other sources. In preliminary studies they identified male DNA in some girls with SLE. Moreover, conducting HLA-specific PCR they found HLA sequences that were specific to an older brother (and not shared with the patient) in patient blood cell-derived DNA. Extending these studies the group is conducting FISH to investigate male cells in kidney biopsies from girls with recent onset SLE. Given their hypothesis (loss of tolerance to non-shared male antigens leads to chronic activation of patient-derived T cells that can attack tissue-residing male chimeric cells), it is interesting to note that HLA-DR15 is more prevalent in girls with SLE. This HLA-DR15 molecule is able to directly present a Y chromosome-derived peptide epitope to female HLA-DR15 restricted CD4 + T cells. Alternatively, HY peptides derived from HLA-DR15neg male cells could be indirectly presented to female CD4 + T cells via HLA-DR15pos antigen-presenting cells from the female host. Collectively, the epidemiological observations presented at the meeting suggest varied and sometimes opposite roles for the various types of naturally acquired Mc in affecting the balance between self-tolerance and autoimmune disease during various stages in life (Fig. 2). The precise role of HLA herein is unclear. Nonetheless, Mc could be seen as a new epigenetic coregulator of autoimmune disease as proposed by Dr Khosrotehrani. The role of Mc in transplantation outcome The 3 speakers addressing this topic, that is Dr Burlingham (University of Wisconsin, Madison, USA), Dr Ichinohe (Hiroshima University, Hiroshima, Japan) and Dr van Halteren
References 1.
2.
3.
4.
5.
6.
Herzenberg LA, Bianchi DW, Schröder J, Cann HM, Iverson GM. Fetal cells in the blood of pregnant women: detection and enrichment by fluorescenceactivated cell sorting. Proc Natl Acad Sci U S A 1979; 76:1453-5; PMID:286330; http://dx.doi. org/10.1073/pnas.76.3.1453 Kroneis T, Gutstein-Abo L, Kofler K, Hartmann M, Hartmann P, Alunni-Fabbroni M, Walcher W, Dohr G, Petek E, Guetta E, et al. Automatic retrieval of single microchimeric cells and verification of identity by on-chip multiplex PCR. J Cell Mol Med 2010; 14:954-69; PMID:19453769; http://dx.doi. org/10.1111/j.1582-4934.2009.00784.x Kroneis T, Geigl JB, El-Heliebi A, Auer M, Ulz P, Schwarzbraun T, Dohr G, Sedlmayr P. Combined molecular genetic and cytogenetic analysis from single cells after isothermal whole-genome amplification. Clin Chem 2011; 57:1032-41; PMID:21558453; http://dx.doi.org/10.1373/clinchem.2011.162131 Choolani M, Mahyuddin AP, Hahn S. The promise of fetal cells in maternal blood. Best Pract Res Clin Obstet Gynaecol 2012; 26:655-67; PMID:22795236; http://dx.doi.org/10.1016/j.bpobgyn.2012.06.008 Dierselhuis MP, Spierings E, Brand R, Hendriks M, Canossi A, Dolstra H, Eliaou JF, Enczmann J, Gervais T, Kircher B, et al. Gender influences the birth order effect in HLA-identical stem cell transplantation. Blood 2013; 121:4809-11; PMID:23744495; http:// dx.doi.org/10.1182/blood-2013-02-485722 Nelson JL. The otherness of self: microchimerism in health and disease. Trends Immunol 2012; 33:4217; PMID:22609148; http://dx.doi.org/10.1016/j. it.2012.03.002
7.
observation that women with unselected breast cancer frequently lack fetal Mc in breast tissue has boosted the concept that these cells can play a protective role in this disease as discussed by Dr Gadi (Fred Hutchinson Cancer Research Center, Seattle, USA).18 Dr. Gadi presented data showing that women with stage 0 breast cancer are similarly deficient in harboring fetal microchimerism in their blood and suggested the timing of the loss of Mc is not a consequence of invasive cancer but a predisposing condition. Although data explaining how Mc of fetal origin could protect against breast cancer is still lacking, Dr Gadi is currently preparing a clinical trial in which stage 4 patients will receive peripheral blood stem cells donated by their offspring. This procedure aims at artificially increasing the levels of Mc of fetal origin in these women. The first results of this exciting next step on exploiting Mc in the management of human disease will hopefully be presented at the next International Symposium on Chimerism. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
We would like to thank all speakers for their input, either during or after the meeting, and suggestions which were of great help composing this manuscript. Daniel Kummer, Amin ElHeliebi, Beate Scheiber, Rudolf Schmied, Manuela Isak, and Evelyn Zötsch are greatly acknowledged for organizing the conference.
Nassar D, Droitcourt C, Mathieu-d’Argent E, Kim MJ, Khosrotehrani K, Aractingi S. Fetal progenitor cells naturally transferred through pregnancy participate in inflammation and angiogenesis during wound healing. FASEB J 2012; 26:14957; PMID:21974929; http://dx.doi.org/10.1096/ fj.11-180695 8. Kara RJ, Bolli P, Karakikes I, Matsunaga I, Tripodi J, Tanweer O, Altman P, Shachter NS, Nakano A, Najfeld V, et al. Fetal cells traffic to injured maternal myocardium and undergo cardiac differentiation. Circ Res 2012; 110:82-93; PMID:22082491; http:// dx.doi.org/10.1161/CIRCRESAHA.111.249037 9. Rak JM, Maestroni L, Balandraud N, Guis S, Boudinet H, Guzian MC, Yan Z, Azzouz D, Auger I, Roudier C, et al. Transfer of the shared epitope through microchimerism in women with rheumatoid arthritis. Arthritis Rheum 2009; 60:7380; PMID:19117368; http://dx.doi.org/10.1002/ art.24224 10. Vanzyl B, Planas R, Ye Y, Foulis A, de Krijger RR, Vives-Pi M, Gillespie KM. Why are levels of maternal microchimerism higher in type 1 diabetes pancreas? Chimerism 2010; 1:45-50; PMID:21327046; http:// dx.doi.org/10.4161/chim.1.2.13891 11. Fugazzola L, Cirello V, Beck-Peccoz P. Microchimerism and endocrine disorders. J Clin Endocrinol Metab 2012; 97:1452-61; PMID:22399520; http://dx.doi.org/10.1210/ jc.2011-3160 12. Lepez T, Vandewoestyne M, Hussain S, Van Nieuwerburgh F, Poppe K, Velkeniers B, Kaufman JM, Deforce D. Fetal microchimeric cells in blood of women with an autoimmune thyroid disease. PLoS One 2011; 6:e29646; PMID:22216337; http:// dx.doi.org/10.1371/journal.pone.0029646
13. Stevens AM, Hermes HM, Kiefer MM, Rutledge JC, Nelson JL. Chimeric maternal cells with tissuespecific antigen expression and morphology are common in infant tissues. Pediatr Dev Pathol 2009; 12:337-46; PMID:18939886; http://dx.doi. org/10.2350/08-07-0499.1 14. Dutta P, Dart M, Roenneburg DA, Torrealba JR, Burlingham WJ. Pretransplant immune-regulation predicts allograft tolerance. Am J Transplant 2011; 11:1296-301; PMID:21449933; http://dx.doi. org/10.1111/j.1600-6143.2011.03484.x 15. van Halteren AG, Jankowska-Gan E, Joosten A, Blokland E, Pool J, Brand A, Burlingham WJ, Goulmy E. Naturally acquired tolerance and sensitization to minor histocompatibility antigens in healthy family members. Blood 2009; 114:226372; PMID:19506299; http://dx.doi.org/10.1182/ blood-2009-01-200410 16. Ichinohe T. Long-term feto-maternal microchimerism revisited: Microchimerism and tolerance in hematopoietic stem cell transplantation. Chimerism 2010; 1:39-43; PMID:21327150; http://dx.doi. org/10.4161/chim.1.1.12743 17. Adams KM, Lambert NC, Heimfeld S, Tylee TS, Pang JM, Erickson TD, Nelson JL. Male DNA in female donor apheresis and CD34-enriched products. Blood 2003; 102:3845-7; PMID:12869496; http:// dx.doi.org/10.1182/blood-2003-05-1570 18. Gadi VK, Nelson JL. Fetal microchimerism in women with breast cancer. Cancer Res 2007; 67:9035-8; PMID:17909006; http://dx.doi.org/10.1158/00085472.CAN-06-4209
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breast feeding, allows ectopic heart transplantation across a major histocompatibility (MHC) barrier in mice. Likewise, mother-tochild haplo-identical stem cell transplantation performed with mother/offspring combinations that are mutually chimeric, seems feasible according to Dr Ichinohe.16 Comparing a large data set of parental donor transplants performed in Japan, maternal donors induced a lower risk of transplantation-associated mortality in their offspring than paternal donors, despite the fact that relapse rates and severe graft-vs.-host disease rates did not differ. Dr. Ichinohe’s current hypothesis is that maternal hematopoietic stem cell grafts may contain Treg specific for the inherited paternal antigens expressed by her offspring. These Treg suppress acute graft-vs.-host responses by maternal alloreactive T cells also present in the graft. According to data presented by Dr van Halteren, this assumption may be true for a proportion of women in whom she detected the pre-transplant presence of HY-specific Treg by tvDTH testing.15 There does not seem to be a clear correlation between the presence of male microchimeric cells, as detected in peripheral blood cells, and HY-specific Treg, however. In line with observations earlier published by Professor Nelson’s research group,17 Dr. van Halteren also presented data on the prevalence of both male mature blood cells and CD34 + progenitor cells in hematopoietic stem cell grafts prepared from G-CSF treated female donors. Whether and how these chimeric cells affect immune responses induced by donor-derived T cells in the female recipients of such grafts is currently investigated by her group. Mc of fetal origin in women with breast cancer Some recent studies have indicated that parity is associated with various pathologic subtypes of breast cancer. The initial
Chimerism 4:4, 132–135; October/November/December 2013; © 2013 Landes Bioscience
Meeting report of the First Symposium on Chimerism Astrid GS van Halteren1,*, Peter Sedlmayr2, Thomas Kroneis2, William J Burlingham3, and J Lee Nelson4 1 Department of Pediatrics; Willem Alexander Children’s Hospital; Leiden University Medical Center; Leiden, the Netherlands; Institute of Cell Biology, Histology and Embryology; University of Graz; Graz, Austria; 3Department of Surgery; Division of Transplantation; University of Wisconsin School of Medicine and Public Health; Madison, WI USA; 4Fred Hutchinson Cancer Research Center; University of Washington; Seattle, WA USA
Keywords: (micro)chimerism, FISH, HLA, PCR, autoimmune disease, cancer, cell trafficking, pregnancy, tissue repair, transplantation
Introduction The incidence and potentially different biological impact of naturally acquired maternal and fetal cells were enthusiastically and extensively discussed when basic scientists and clinicians met at the Medical University of Graz in Austria for an International Symposium on Chimerism (Fig. 1). The two day program featured presentations by speakers from 4 different continents, intermingled with short presentations focusing on technical aspects of chimerism analysis by representatives from the companies who sponsored the meeting. Details on the program and speakers list are available at the meeting’s website (http:// chimerism.medunigraz.at/cms/website.php). As also exemplified by the various different types of studies published in the recently released journal Chimerism, this field is slowly but steadily expanding as new research teams working outside and within the transplantation field have become interested in the biologically intriguing phenomena of chimerism and microchimerism (Mc). The highlights of the meeting are summarized in this meeting report.
Technical Aspects of Chimerism Analysis Since the 1979 paper by Herzenberg and colleagues reporting enrichment of cells from pregnant women presumed to be fetal using flow-sorting techniques,1 many different studies have been published describing fetal or maternal cells in diverse hematopoietic cell types or tissues and organs. During the past decade, major technical improvements regarding the detection of these generally rare cells have been made. This has resulted in a shift from the more traditional XY fluorescent in situ hybridization (FISH) staining technique applied to tissue sections to more refined technologies such as real-time PCR for the detection of male-specific DNA sequences in genomic DNA extracted from peripheral blood cells or processed tissues. Realtime PCR reactions for mismatched HLA alleles as well as for other genetic markers (insertion/deletion) have been developed *Correspondence to: Astrid GS van Halteren; Email: [email protected] Submitted: 11/11/2013; Accepted: 11/12/2013 http://dx.doi.org/10.4161/chim.27168
in the laboratories of meeting participants Dr Nelson (Fred Hutchinson Cancer Research Center, Seattle, USA), Dr Lambert (INSERM UMRs 1097, Marseille, France) and Dr van Halteren (Leiden University Medical Center, Leiden, the Netherlands). Unfortunately, many researchers are faced by the problem that this approach requires the availability of DNA from mother or offspring in order to find informative genetic markers. Of note, the newly developed QuantiChimera platform established at the laboratory of Dr Lambert can be contacted for technical support regarding state-of-the art PCR technology for Mc detection. Particularly relevant for investigators who are restricted to using FISH techniques for the identification of rare maternal or fetal chimeric cells is the collective observation that this technique does not always give consistent staining results. This may be explained by the fact that archived tissue samples are often subjected to different fixation protocols. As discussed by Dr Sedlmayr and Dr Kroneis (Medical University, Graz, Austria), FISH by itself is a less than optimal technique for the analysis of rare microchimeric cells given the frequency of false positive cells. This issue can in part be addressed by using two reverse sets of differently labeled X and Y probes. Also biochemical markers such as unique cell surface molecules or markers that can be used to distinguish fetal cells from adult cells are not perfectly reliable in the setting of analysis of extremely rare cells. Unambiguous identification of individual microchimeric cells can be done by combining either FISH analysis or analysis of biochemical markers with DNA genotyping of candidate cells isolated by laser microdissection.2 The introduction of a whole genome amplification step subsequently even allows combining DNA genotyping of single microchimeric cells with sequencing or comparative genome hybridization.3 This exciting new approach is expected to further boost the chimerism field.
Why Our Natural Immigrants Matter The main route of acquiring chimeric cells from mother and/or fetus is placental exchange of cells during pregnancy. As presented by Dr Hahn (University Hospital Basel, Basel, Switzerland) fetal cell trafficking to the mother starts already very early in pregnancy and is significantly increased in women with placental defects including preeclampsia.4 Using HLA-specific PCR, several groups have now collected evidence strongly suggesting
132 Chimerism
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©2013 Landes Bioscience. Do not distribute.
2
Figure 1.The Symposium on Chimerism. Photo courtesy of Harry Schiffer, Copyright Graz Tourism.
that male cells can also be acquired from an older brother (transmaternal cell trafficking)5 or even from a twin brother who died during embryonic development (“vanished” twin). As discussed by Dr Nelson, particularly adult women may bear chimeric blood cells from multiple sources6. Intriguingly, maternal cells acquired during fetal/neonatal life seem to be replaced by fetal-derived cells when females enter the reproductive phase. Alternatively, pregnancy in later life may induce redistribution of maternal cells in tissues. As outlined in Figure 2, these naturally acquired fetal or maternal cells may affect normal biological processes such as tissue repair and reproduction, but also seem to play a role in an array of pathological responses occurring later in life; the latter include disease features associated with autoimmunity, infection, transplantation and cancer. These topics were addressed at the meeting. Tissue repair Mechanistic studies on chimeric cell trafficking and function in experimental animal models are now feasible due to the generation of green fluorescent protein (GFP) reporter mice, which enable researchers to visualize and study the biological impact of fetal-maternal and maternal-fetal cell trafficking in vivo. Mouse studies on fetal cell contribution to tissue repair were nicely presented by Dr Khosrotehrani (University of Queensland, Brisbane, Australia) and Dr Chaudhry (Mount Sinai School of Medicine, New York, USA). Dr Khosrotehrani’s research focuses on fetal cell trafficking to experimentally induced inflammation to tissues or organs.7 Using bioluminescence imaging, data from his group showed that GFP-tagged fetal mesenchymal cells acquired by female mice through pregnancy are recruited to experimentally injured skin and kidney tissue where they respectively produce collagen and induce angiogenesis of small blood vessels comprised of maternal and fetal endothelial cells. Dr Chaudhry’s group is studying fetal cell migration and in situ differentiation of these cells to understand the high recovery rates of peripartum cardiomyopathy, an inflammatory condition which may occur during the last stage of pregnancy up to 6 mo after delivery.8 Using GFP-reporter mice, she elegantly showed that cells originating from the fetus home to the hearts of female mice with experimentally induced myocardial injury.
Meeting Report
These cells express various types of proteins which are typically expressed by cardiomyocytes. Furthermore, GFP+ cells isolated from maternal hearts form vascular tubes and even form beating cardiomyocytes when cultured on feeder layers. Remarkably, a significant percentage of fetal cells present in maternal hearts express transcription factors such as Cdx2 which is typically expressed by placental stem cells (trophoblast stem cells). Whether placenta-derived Cdx2 expressing cells can be used in cardiovascular regenerative therapy is currently under investigation. Microchimerism in autoimmune disease Mc, identified in peripheral blood and tissues, has been strongly implicated in systemic sclerosis.6 As presented by Dr Lambert, genome-wide association studies have revealed that HLA-DRB1*1104 is associated with the highest risk of scleroderma in Caucasian populations. One of the still unsolved issues is the observation that women who have given birth to an HLA-compatible child are at an increased risk of developing systemic sclerosis. This observation opened the discussion whether HLA compatibility in several generations of one family facilitates the establishment or expansion of Mc. A large study is needed to address this issue. In addition, it has been suggested that Mc may have a “mini-gene transfer” like effect, conferring risk or protection from a disease depending on the particular HLA allele carried by the Mc,9 as discussed by Dr Lambert and colleagues. Another unsolved question is whether or not the same microchimeric cells exert different biological effects. Microchimeric cells, either from fetal or maternal origin, can be seen as circulating or tissue-residing targets for host-derived pathological T cells resulting in autoimmune inflammation. This possibility was discussed by D. Gillespie (University of Bristol, Bristol, UK) who presented data demonstrating that maternal Mc in peripheral blood, as measured by real-time PCR specific for non-inherited maternal HLA sequences, is increased in children with type 1 diabetes. Combining XY FISH and immunostaining on pancreatic tissue from individuals with type 1 diabetes and healthy controls, she showed that tissue-residing maternal cells express tissue-specific markers including insulin (marking β cells) or glucagon (marking α cells) but CD45+ve cells in the insulitic infiltrate were not maternal.10 These results suggest that pancreas-residing, partially HLA mismatched, chimeric cells are not the effectors of the autoimmune destruction process, but rather perhaps are involved in the initiation of autoimmunity by triggering maternal alloreactive T cells. Alternatively, chimeric cells may facilitate the repair of damaged cells in the islets of Langerhans. These maternal microchimeric cells have been observed clustered in small sections of the islets. Whether this pattern somehow prevents destruction of all insulin-producing β cells remains speculative. Another interesting discussion point as raised by Dr Hahn is the possibility that the maternal Mc carry a virus which may trigger the onset of insulitis. Mc can somehow decrease the risk of autoimmune disease. This dual action is best exemplified in the setting of Rheumatoid Arthritis (RA) as discussed by Drs Lambert and Nelson. Women who lack RA susceptibility HLA alleles9 (encoding QKRAA
www.landesbioscience.com Chimerism 133
©2013 Landes Bioscience. Do not distribute.
Meeting Report
Figure 2. The Jing Jang of naturally acquired microchimerism, some examples of fetal and maternal microchimerism that were discussed. Fetal or maternal microchimeric cells may positively affect normal biological responses and/or protect the person who harbors the microchimerism from pathological challenges. On the other hand, microchimerism may also play a negative role by directly or indirectly contributing to immunopathology.
(Leiden University Medical Center, Leiden, the Netherlands), all presented data favoring the concept that microchimeric cells can have a profound effect on the host’s alloreactive T cell repertoire. Dr Burlingham proposed a new term for alloantigens presented by microchimeric cells: Naturally Acquired Chimerism-Derived Occult Antigens, NACHOs. Using carefully selected mouse breeding models, work from his group has clearly demonstrated that NACHOs-expressing maternal microchimeric cells can be found in peripheral blood, bone marrow (c-kit+ progenitor cells) and in various organs such as the heart. The percentage of organs expressing maternal Mc correlates with the degree of immune regulation to non-inherited maternal antigens (NIMA) expressed by these chimeric cells. Surprisingly, they found that maternal Mc was difficult to detect in lymphoid organs such as spleen, lymph nodes, and thymus. However spleen, lymph node and blood contained a relatively high proportion of host MHC class II expressing cells that had acquired maternal antigens. This NIMA-specific antigen acquisition may provide the missing link between Mc and tolerance to a subsequent transplant. T cells that mediate tolerance to NIMA+ allografts can be detected by transvivo delayed type hypersensitivity (tvDTH) testing. This assay works well for analyzing the prevalence and function of both murine14 and human15 T regulator cells (Treg). An instruction video on the principles of this assay can be seen at the following website: http://www.jove.com/video/4454/trans-vivo-delayedtype-hypersensitivity-assay-for-antigen-specific. As presented by Dr Burlingham, the pre-transplant existence of NIMA tolerance, as indicated by the presence of tissueresident maternal Mc acquired during fetal development and
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or QRRAA sequences) can acquire Mc that encodes RA susceptibility alleles and this type of Mc is thought to increase RA risk. Other studies suggest, on the other hand, that if Mc encoding a HLA sequence which is protective against RA (i.e., DERAA encoding HLA alleles) is acquired, the presence of such cells may decrease the risk of developing RA. In both cases, microchimeric cells could be acquired from an individual’s mother or, in women, from the woman’s own prior pregnancies. Chimeric cells acquired from the mother during fetal life occur at a phase when the immune system is still developing. In contrast, fetal cells exchanged during pregnancy are encountered by a fully matured maternal immune system. Whether or not these two sources of Mc indeed differ remains a question of interest for further studies. As discussed by Dr Fugazzola (University of Milan, Milan, Italy), fetal Mc is also detected at higher frequency in autoimmune thyroid disease (Graves disease and Hashimoto thyroiditis) compared with non-autoimmune disease-affected thyroid tissues and post-mortem analyzed normal thyroid glands.11 The limited data available on fetal Mc in autoimmune thyroid diseases at the peripheral blood level seem to indicate its pathogenic role.12 In contrast to earlier studies on this topic, data from the Milan group showed a prevalence of circulating fetal Mc that was actually lower in women with Graves disease as compared with healthy controls. This observation suggests there may be more complexity to a possible role of fetal Mc in Graves’ disease. Dr Stevens (University of Washington, Seattle, USA) presented data on the role of Mc in children with systemic lupus erythematosus (SLE). Her group is studying maternal Mc13 as well as Mc originating from other sources. In preliminary studies they identified male DNA in some girls with SLE. Moreover, conducting HLA-specific PCR they found HLA sequences that were specific to an older brother (and not shared with the patient) in patient blood cell-derived DNA. Extending these studies the group is conducting FISH to investigate male cells in kidney biopsies from girls with recent onset SLE. Given their hypothesis (loss of tolerance to non-shared male antigens leads to chronic activation of patient-derived T cells that can attack tissue-residing male chimeric cells), it is interesting to note that HLA-DR15 is more prevalent in girls with SLE. This HLA-DR15 molecule is able to directly present a Y chromosome-derived peptide epitope to female HLA-DR15 restricted CD4 + T cells. Alternatively, HY peptides derived from HLA-DR15neg male cells could be indirectly presented to female CD4 + T cells via HLA-DR15pos antigen-presenting cells from the female host. Collectively, the epidemiological observations presented at the meeting suggest varied and sometimes opposite roles for the various types of naturally acquired Mc in affecting the balance between self-tolerance and autoimmune disease during various stages in life (Fig. 2). The precise role of HLA herein is unclear. Nonetheless, Mc could be seen as a new epigenetic coregulator of autoimmune disease as proposed by Dr Khosrotehrani. The role of Mc in transplantation outcome The 3 speakers addressing this topic, that is Dr Burlingham (University of Wisconsin, Madison, USA), Dr Ichinohe (Hiroshima University, Hiroshima, Japan) and Dr van Halteren
References 1.
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Herzenberg LA, Bianchi DW, Schröder J, Cann HM, Iverson GM. Fetal cells in the blood of pregnant women: detection and enrichment by fluorescenceactivated cell sorting. Proc Natl Acad Sci U S A 1979; 76:1453-5; PMID:286330; http://dx.doi. org/10.1073/pnas.76.3.1453 Kroneis T, Gutstein-Abo L, Kofler K, Hartmann M, Hartmann P, Alunni-Fabbroni M, Walcher W, Dohr G, Petek E, Guetta E, et al. Automatic retrieval of single microchimeric cells and verification of identity by on-chip multiplex PCR. J Cell Mol Med 2010; 14:954-69; PMID:19453769; http://dx.doi. org/10.1111/j.1582-4934.2009.00784.x Kroneis T, Geigl JB, El-Heliebi A, Auer M, Ulz P, Schwarzbraun T, Dohr G, Sedlmayr P. Combined molecular genetic and cytogenetic analysis from single cells after isothermal whole-genome amplification. Clin Chem 2011; 57:1032-41; PMID:21558453; http://dx.doi.org/10.1373/clinchem.2011.162131 Choolani M, Mahyuddin AP, Hahn S. The promise of fetal cells in maternal blood. Best Pract Res Clin Obstet Gynaecol 2012; 26:655-67; PMID:22795236; http://dx.doi.org/10.1016/j.bpobgyn.2012.06.008 Dierselhuis MP, Spierings E, Brand R, Hendriks M, Canossi A, Dolstra H, Eliaou JF, Enczmann J, Gervais T, Kircher B, et al. Gender influences the birth order effect in HLA-identical stem cell transplantation. Blood 2013; 121:4809-11; PMID:23744495; http:// dx.doi.org/10.1182/blood-2013-02-485722 Nelson JL. The otherness of self: microchimerism in health and disease. Trends Immunol 2012; 33:4217; PMID:22609148; http://dx.doi.org/10.1016/j. it.2012.03.002
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observation that women with unselected breast cancer frequently lack fetal Mc in breast tissue has boosted the concept that these cells can play a protective role in this disease as discussed by Dr Gadi (Fred Hutchinson Cancer Research Center, Seattle, USA).18 Dr. Gadi presented data showing that women with stage 0 breast cancer are similarly deficient in harboring fetal microchimerism in their blood and suggested the timing of the loss of Mc is not a consequence of invasive cancer but a predisposing condition. Although data explaining how Mc of fetal origin could protect against breast cancer is still lacking, Dr Gadi is currently preparing a clinical trial in which stage 4 patients will receive peripheral blood stem cells donated by their offspring. This procedure aims at artificially increasing the levels of Mc of fetal origin in these women. The first results of this exciting next step on exploiting Mc in the management of human disease will hopefully be presented at the next International Symposium on Chimerism. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
We would like to thank all speakers for their input, either during or after the meeting, and suggestions which were of great help composing this manuscript. Daniel Kummer, Amin ElHeliebi, Beate Scheiber, Rudolf Schmied, Manuela Isak, and Evelyn Zötsch are greatly acknowledged for organizing the conference.
Nassar D, Droitcourt C, Mathieu-d’Argent E, Kim MJ, Khosrotehrani K, Aractingi S. Fetal progenitor cells naturally transferred through pregnancy participate in inflammation and angiogenesis during wound healing. FASEB J 2012; 26:14957; PMID:21974929; http://dx.doi.org/10.1096/ fj.11-180695 8. Kara RJ, Bolli P, Karakikes I, Matsunaga I, Tripodi J, Tanweer O, Altman P, Shachter NS, Nakano A, Najfeld V, et al. Fetal cells traffic to injured maternal myocardium and undergo cardiac differentiation. Circ Res 2012; 110:82-93; PMID:22082491; http:// dx.doi.org/10.1161/CIRCRESAHA.111.249037 9. Rak JM, Maestroni L, Balandraud N, Guis S, Boudinet H, Guzian MC, Yan Z, Azzouz D, Auger I, Roudier C, et al. Transfer of the shared epitope through microchimerism in women with rheumatoid arthritis. Arthritis Rheum 2009; 60:7380; PMID:19117368; http://dx.doi.org/10.1002/ art.24224 10. Vanzyl B, Planas R, Ye Y, Foulis A, de Krijger RR, Vives-Pi M, Gillespie KM. Why are levels of maternal microchimerism higher in type 1 diabetes pancreas? Chimerism 2010; 1:45-50; PMID:21327046; http:// dx.doi.org/10.4161/chim.1.2.13891 11. Fugazzola L, Cirello V, Beck-Peccoz P. Microchimerism and endocrine disorders. J Clin Endocrinol Metab 2012; 97:1452-61; PMID:22399520; http://dx.doi.org/10.1210/ jc.2011-3160 12. Lepez T, Vandewoestyne M, Hussain S, Van Nieuwerburgh F, Poppe K, Velkeniers B, Kaufman JM, Deforce D. Fetal microchimeric cells in blood of women with an autoimmune thyroid disease. PLoS One 2011; 6:e29646; PMID:22216337; http:// dx.doi.org/10.1371/journal.pone.0029646
13. Stevens AM, Hermes HM, Kiefer MM, Rutledge JC, Nelson JL. Chimeric maternal cells with tissuespecific antigen expression and morphology are common in infant tissues. Pediatr Dev Pathol 2009; 12:337-46; PMID:18939886; http://dx.doi. org/10.2350/08-07-0499.1 14. Dutta P, Dart M, Roenneburg DA, Torrealba JR, Burlingham WJ. Pretransplant immune-regulation predicts allograft tolerance. Am J Transplant 2011; 11:1296-301; PMID:21449933; http://dx.doi. org/10.1111/j.1600-6143.2011.03484.x 15. van Halteren AG, Jankowska-Gan E, Joosten A, Blokland E, Pool J, Brand A, Burlingham WJ, Goulmy E. Naturally acquired tolerance and sensitization to minor histocompatibility antigens in healthy family members. Blood 2009; 114:226372; PMID:19506299; http://dx.doi.org/10.1182/ blood-2009-01-200410 16. Ichinohe T. Long-term feto-maternal microchimerism revisited: Microchimerism and tolerance in hematopoietic stem cell transplantation. Chimerism 2010; 1:39-43; PMID:21327150; http://dx.doi. org/10.4161/chim.1.1.12743 17. Adams KM, Lambert NC, Heimfeld S, Tylee TS, Pang JM, Erickson TD, Nelson JL. Male DNA in female donor apheresis and CD34-enriched products. Blood 2003; 102:3845-7; PMID:12869496; http:// dx.doi.org/10.1182/blood-2003-05-1570 18. Gadi VK, Nelson JL. Fetal microchimerism in women with breast cancer. Cancer Res 2007; 67:9035-8; PMID:17909006; http://dx.doi.org/10.1158/00085472.CAN-06-4209
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breast feeding, allows ectopic heart transplantation across a major histocompatibility (MHC) barrier in mice. Likewise, mother-tochild haplo-identical stem cell transplantation performed with mother/offspring combinations that are mutually chimeric, seems feasible according to Dr Ichinohe.16 Comparing a large data set of parental donor transplants performed in Japan, maternal donors induced a lower risk of transplantation-associated mortality in their offspring than paternal donors, despite the fact that relapse rates and severe graft-vs.-host disease rates did not differ. Dr. Ichinohe’s current hypothesis is that maternal hematopoietic stem cell grafts may contain Treg specific for the inherited paternal antigens expressed by her offspring. These Treg suppress acute graft-vs.-host responses by maternal alloreactive T cells also present in the graft. According to data presented by Dr van Halteren, this assumption may be true for a proportion of women in whom she detected the pre-transplant presence of HY-specific Treg by tvDTH testing.15 There does not seem to be a clear correlation between the presence of male microchimeric cells, as detected in peripheral blood cells, and HY-specific Treg, however. In line with observations earlier published by Professor Nelson’s research group,17 Dr. van Halteren also presented data on the prevalence of both male mature blood cells and CD34 + progenitor cells in hematopoietic stem cell grafts prepared from G-CSF treated female donors. Whether and how these chimeric cells affect immune responses induced by donor-derived T cells in the female recipients of such grafts is currently investigated by her group. Mc of fetal origin in women with breast cancer Some recent studies have indicated that parity is associated with various pathologic subtypes of breast cancer. The initial
