RESEARCH PAPER Chimerism 5:3-4, 80--85; July–December 2014; © 2014 Taylor & Francis Group, LLC
Microchimerism and regulation in living related kidney transplant families W John Haynes, Ewa Jankowska-Gan, Lynn Haynes, and William J Burlingham* Department of Surgery; School of Medicine and Public Health; University of Wisconsin; Madison, WI USA
Long-term harmful effects of immunosuppressive drugs and chronic rejection are a persistent impetus to establish methods to induce immunological tolerance to allografts. PCR-based studies have found evidence that humans and other placental mammals can have prolonged extremely low levels of maternal cells as well as other non-self cells, referred to as microchimerism. The persistence of these cells suggests a mechanism for the maintenance of the regulatory T-cell (Treg) responses frequently detected in offspring to non-inherited maternal antigens. We test the hypothesis that the detection of very low copy levels of insertion/deletion (Indel) alleles consistent with non-inherited maternal genes, will correlate with immune regulation to non-inherited maternal antigens as detected by a trans-vivo Delayed-Type Hypersensitivity (tvDTH) assay in kidney transplant recipients, normal donors and their immediate biological family members. Preliminary data reported here compares qPCR amplification of rare DNA templates in the peripheral blood polymorphonuclear (PMN) fraction of cells, with the results of tvDTH assays for linked suppression of recall antigen responses in the presence of non-inherited maternal antigens [NIMA]. The two assays do not show a definitive correlation.
Introduction The goal of transplanted organs providing a cure for a variety of diseases is limited by the long-term chronic toxicity of the immunosuppressive drugs currently required to regulate the immune response to non-self antigens and maintain good graft function.1 Efforts to reduce the toxicity have led to new drugs and different maintenance therapies.2 However the hope of eliminating the need for these drugs requires expanding our understanding of how several mechanisms (anergy, clonal deletion and regulation) establish tolerance to non-self antigens. The earliest publication of natural tolerance in mammals was the description of freemartin cattle by Ray Owen in 1945.3 Owen later reported the first evidence for acquired tolerance to non-inherited maternal antigens (NIMA) in humans.4 Since then numerous studies have attempted to characterize the details of the mechanism(s) involved in the tolerance to NIMA.5-10 In mice, backcrossing a maternal hybrid [C57BL/6 (H2b) x DBA/2 (H2d)] that is heterozygous at the MHC locus (i.e. H2) with a homozygous (b\b) male resulted in half of the (b\b) offspring able to accept a fully allogeneic heart (d\d) without immunosuppresion.7 This capacity was strongly correlated with T reg activity detected pre-transplant and long-term persistence of a maternal allele indirectly measured by late cycle PCR.11 The persistence of the maternal microchimerism was curiously dependent on exposure to the maternal antigens during nursing and maybe related to the maturation of the mouse immune system after birth.11 The proposed theory was that maintenance of the NIMA-specific TGFbC CD4+ Treg cells required continuous
exposure to the antigen provided by persistent maternal microchimerism in several organs as well as in the bone marrow.8,12 Recent intricate experiments have provided striking evidence that an adult female, exposed in utero to a transgenic NIMA, when treated with an antibody against the same transgenic NIMA rapidly loose Treg cells specific for the NIMA construct.13 The inference is that elimination of cells expressing the antigen negatively impacts the maintenance of Treg to those antigens. More interestingly, in adult females there was a dramatic impact on the ability to maintain a pregnancy when the Tregs to antigens expressed by the fetus were eliminated. In humans, the trans-vivo delayed-type hypersensitivity assay (tvDTH) is one of the very few assays that measures regulation to specific antigens and has continued to provide evidence for NIMA regulation.14-16 The assay places cells from humans into the footpad of a severe combined immunodeficient (SCID) mouse along with a known potent recall antigen (e.g. tetanus). This elicits a swelling response dependent on the activation of established memory-effector T-cells by way of appropriate antigen presenting cells in the injected cell population. In specific cases, the injection of additional secondary antigens decreases (or rapidly resolves) the swelling response.15 This reduced swelling is referred to as linked suppression (or regulation) and requires both dendritic cells and antigen-specific T regulatory cells. This assay has revealed that cells from the peripheral blood of offspring very frequently show a regulated response to non-inherited maternal antigens in humans [the NIMA effect17] The fact that large prospective studies of outcomes among human living related kidney transplant populations do not always show a clear
*Correspondence to: William J Burlingham; Email: [email protected] Submitted: 06/25/2015; Revised: 09/15/2015; Accepted: 10/16/2015 http://dx.doi.org/10.1080/19381956.2015.1111974
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beneficial effect of maternal over paternal mismatched kidney transplants (NIMA vs. NIPA), demonstrate that other factors must be involved.18,19 One such factor is the response of the donor to the recipient. It has been shown that between mothers and their offspring, a mutual or bi-directional regulation appears to be of clear benefit in living related kidney transplants.17,20 In this report we present data to test the hypothesis that there is a correlation between regulation to non-inherited maternal antigens among family members and evidence for persistent microchimeric maternal alleles indirectly detected by PCR. The tvDTH assay requires the separation of peripheral blood mononuclear cells (PBMC) from a very abundant polymorphonuclear (PMN) cell fraction. The significant numbers of PBMC used in the tvDTH assay often limited the number of cells that could be routinely used to extract total DNA template for qPCR, whereas the PMN fraction was often discarded. The PMN fraction typically also contains about 10% lymphocytes, and therefor is a readily available, abundant pool of peripheral blood cells that could be routinely used to extract DNA. The majority of the haematopoietic cells in the bone marrow develop into myeloid cells and specifically into mature neutrophils. This population of cells has a very short half-life in the peripheral blood (8-12 hours to perhaps up to 5 days or longer),21 the cells continuously egress from the bone marrow and a previous publication has reported detecting maternal microchimerism in this cell fraction.22 These cells therefor should reflect a continuously renewed sampling of haematopoietic precursor cells from the bone marrow and provide indirect evidence for maternal cells in the bone marrow.
Material and Methods Study participants Immunological monitoring was performed on samples obtained from healthy family members and end stage renal disease (ESRD) patients according to informed consent procedures, subject to human subjects Institutional Review Board approval at the University of Wisconsin-Madison. Isolation of leukocyte cell types Peripheral blood mononuclear cells (PBMC) used in the tvDTH were the entire mixed “buffy coat” of cells (T-cells, B-cells, monocytes, and a significant frequency of contaminating neutrophils) isolated by density gradient centrifugation using a sterile, iso-osmotic polysucrose and diatrizoate solution (Lymphocyte Separation Medium; Corning). Cells for chimerism testing were obtained from the remaining fraction of cells after lysing red blood cells with NH4Cl and KHCO3 lysis (ACK) buffer. The majority of the cells in this fraction were polymorphonuclear (PMN; a.k.a. neutrophils) with an average of about 10% mononuclear lymphocytes. Testing Treg function in the trans vivo Delayed Type Hypersensitivity assay Fresh human PBMC [7–9£106] in PBS were mixed with cell sonicate-derived antigen prepared from PBMC and injected into the footpad of 6-8 week old CB.17 SCID mice, as described
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previously.14,15,23 The response to tetanus toxoid recall antigen alone plus PBMC was used as a positive control, with PBMC alone as a negative control. To test for bystander suppression, a recall antigen was co-injected with test antigen (antigen from family members). Footpad thickness was measured using a spring-loaded caliper at time 0 (before injection) and 24 hours after injection. Net swelling was determined by subtracting “background” swelling from a control injection of PBMC alone. The extent of bystander suppression was measured as % inhibition of the recall antigen response in the presence of test antigen, calculated using the following formula: % Inhibition (or % Regulation) D [1-(recall+test antigen)/ recall alone] x 100% Total DNA isolation Each sample processed contained 2£107 cells from the PMN fraction from the same peripheral blood samples used in the tvDTH assays. Purification was done using QIAmp DNA blood minikit (QIAGEN) with minor modifications. An RNase cocktail (Ambion) was used to insure the removal of all RNA from the sample. During the 56 C incubation used to digest protein and RNA, a syringe was used to disperse cellular material to facilitate digestion. After addition of Ethanol, repeated pipetting along the side of the tube was done to facilitate fragmenting DNA. Experiments determined that column purification does not work efficiently for fragments larger than 25 Kb and fragments 50 Kb or larger do not elute from the columns (data not shown). The dispersion during digestion and the fragmentation by pipetting increased the average yield 2-4 fold. Total nucleic acid concentration of each sample was quantified on a NanoDrop 1000 spectrophotometer (Thermo Scientific). Genomic DNA fragments in samples were examined using intercalating Ethidium Bromide after separation by 0.7% agarose gel electrophoresis to ensure fragmentation was minimal and all RNA had been removed. All PCR products were also examined and validated by 2.0% agarose gel electrophoresis. Detection of microchimeric template variation A set of previously published primers and probes were used to distinguish insertion deletion alleles that maintain a high degree of heterozygosity at 8 somatic loci and one X-linked locus in humans.24,25 In addition, primers and probes for 3 loci on the male chromosome were used to test if the frequency of male microchimerism was similar to previous reports.26-29 The DAZ (DYS1) primers and TSPY (DYS14) primers amplify 2 different polyploid sequences on the Y chromosome. The actual copy number appears to be somewhat variable.29,30 The TSPY primers and probe used here are newly designed shifting the amplicon (a PCR amplified product) 75 bases from the original DYS14 primers first used by Lo et al. and later by Lambert et al.26,31 (sequence available upon request). The DAZ primers were the same as previously described.32,33 The SRY primers amplify a single copy locus and along with the GAPDH positive control were described by Johnson et al.28 Both SYBR green (SABioscience and later Qiagen) and probe-based (SsoFast; BioRad) qPCR chemistry were used in several replicate reactions. Patients
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and family members were screened to determine the alleles inherited at a normal complement level. The screen was generally done at a minimum of 2 concentrations of template (neet and diluted to either 20, 50 or 100ng/ml). Template was also tested after digestion with the restriction enzyme HinDIII, known not to cut any of the amplicons, to ensure maximum amplification efficiency was tested. Efforts to use high concentrations (>20 ng/ ml of reaction) proved to inhibit amplification and frequently reduced the sensitivity of the assays. The condition used in general for the screen reported here were multiple 10ml reactions held at 95 C for 5 minutes, then cycled from 95 C for 5 seconds to 60 or 62 C annealing and extension for 15 seconds in a quick 2 step PCR for 50 cycles. Dilutions of positive template into negative template demonstrated that the assays were generally sensitive enough to amplify as little as a few and sometimes as little as one copy of target loci per tube. Results QPCR analysis of microchimerism The cohort reported here includes 16 mothers, 4 fathers, and 32 offspring for a total of 52 individuals examined by qPCR at 12 loci, including 15 ESRD patients and their paired normal donors (Table 1). The male loci tested have varying degrees of natural ploidy along the Y chromosome and were used to assess whether detection of male microchimerism correlated with the expected copy number variation and other published data. The results of the screen for male microchimerism found 41% and 38% late cycle amplicons in the 2 polyploid loci (DAZ, TSPY) and 21% at the single copy locus (SRY; Table 1). The efficiency of amplification of the 2 different polyploid loci amplicons did not result in significant difference in the detection of male microchimerism. The overall frequency of male microchimerism found is similar to the frequency previously reported.16,31 The insertion/deletion (InDel) loci heterozygosity in this cohort of 52 individuals was between 43 and 67% (Table 1). Control reactions without template typically yielded no products
except for infrequent primer dimers or accidental contamination from a neighboring well. These events were extremely low frequency, had very late amplification with distinctly different amplification efficiencies. Templates from many individuals that did not inherit one of the alleles routinely had no amplification or infrequently distinctly incorrect alternate products, again with different amplification efficiencies and amplicon lengths. Every reaction was examined by both gel electrophoresis and second derivative melting curves. In contrast, templates from some individuals routinely generated distinctly late amplifying products of the expected size (Fig. 1A). These low copy alleles on average appeared with a ΔCt of between 10 and 15 compared with both the alternate allele and GAPDH as normal genetic complement controls. The late cycle amplicons, generally, amplified earlier than other types of false positive (primer dimers, etc.) and had amplification efficiencies similar to true positive amplicons (Fig. 1B). Each negative allele showing signs of late amplifying product were re-examined by replicate qPCR in comparison with template that was a consistent negative for the allele and a second template with consistent positive normal complement allele. This particular example is an ESRD patient and the living related donor. The ESRD patients and normal living donors showed no significant difference in the frequency of late cycle (low copy) amplification of products (ND15; 98 and 104 low copy or negative alleles tested, respectively). Screening this population for testable NIMA loci, a total of 23 individual offspring were available to examine for potential noninherited maternal late cycle amplicons and correlate with the regulation assay (Fig. 2). 5 females (D) and 6 males ( ) show no evidence of late amplifying non-inherited maternal amplicons (No NIMA Mc). 4 individuals in this group had late amplification of alleles that were not NIMA (red outline; either microchimerism in alleles not carried by mother or male microchimerism). In the second group there were 12 individuals that had at least one allele with a late cycle NIMA amplicon, and some individuals also had late cycle nonNIMA amplicons. There was clearly no statistically significant difference between these 2 groups with respect to regulation using either unpaired T-test or a Mann-Whitney non-parametric test (p>0.5).
Table 1. Summary statistics of the PCR for 52 individuals. 18 males and 34 women (Median age: 54; minimum age: 21). 16 of the women had late cycle male amplicons at either the polyploid loci DAZ, or TSPY, and in some cases the single locus SRY (Row 3, first 3 columns). The polyploid loci were consistently detected at an earlier cycle than the single locus SRY, but in some women only the polyploid loci were amplified. The remaining somatic InDel loci were generally of high heterozygosity (Row 7) and could be frequently interrogated for low copy templates (row 8) and despite differences in reaction kinetics showed a similar average ΔCq with the reference GAPDH (row 9). Male Loci DAZ TSPY SRY 1) positive 2) negative 3) late cycle amplicon 4) Total negative+late 5) Total defined alleles 6) % negative or late 7) Heterozyogousity 8) % of negative w/late amplicon 9) Average DCq (Loci-GAPDH)
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S01 del
S04 in
del
18 18 18 39 42 37 18 20 27 12 9 9 16 14 7 1 1 6 34 34 34 13 10 15 52 52 52 52 52 52 65% 65% 65% 25% 19% 29% — — — 44.2% 42.3% 47% 41% 21% 8% 10% 40% 14 13 18 12 15 14
S07 in S06
del
45 7 0 7 52 13%
28 40 20 9 4 3 24 12 52 52 46% 23% 67.3% 0% 17% 25% — 16 15
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S08 in
del
29 44 18 2 5 6 23 8 52 52 44% 15% 42.3% 22% 75% 15 14
S09 in
38 8 6 14 52 27%
del
49 1 2 3 52 6% 65.4% 43% 67% 13 15
S10 in
del
21 40 26 9 5 3 31 12 52 52 60% 23% 46.2% 16% 25% 14 13
S11 in
del
Tg in
38 33 12 14 2 5 14 19 52 52 27% 37% 46.2% 14% 26% 19 15
del
47 31 5 16 0 5 5 21 52 52 10% 40% 57.7% 0% 24% — 16
in 43 8 1 9 52 17% 11% —
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Figure 1. Example of a late cycle amplification. (A) Early amplification of a heterozygous template (donor; 298.2 ng/10ml; gel lane 2) for both S08 insertion and deletion alleles compared with a template homozygous for S08 insertion (recipient; 292 ng/10 ml; gel lane 4) with a late amplifying S08 deletion (recipient; gel lane 3) amplicon. A positive GAPDH control for both templates (gel lane 5). (B) Overlapping derivative melt curves for 3 products S08 insertion, S08 deletion and GAPDH show slight shifts in peak melting temperature.
Discussion The data indicates no clear relationship between the late cycle amplification products from the PMN fraction and regulation to NIMA as measured by the tvDTH assay. This is a very similar result to one recently published looking for a correlation of male microchimerism with regulation to minor HY antigens in a variety of cell fractions emphasizing different cell types.16 In this report, the clear and repeatable detection of late cycle amplicons in specific templates substantiates the usefulness of the InDel primers and the PMN fraction in looking for alternate low copy alleles. The PMN fraction provides a very large numbers of cells that can be examined while leaving the PBMC for tvDTH or other regulation assays. In addition, the tendency for these cells to maintain more diffuse, less compact nuclei allows DNA to be more efficiently purified and ready for PCR amplification (personal observation). The challenge in making strong conclusions from the PCR data presented here is that there are always improbable but potential reasons for late cycle amplification from the expected locus with a
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Figure 2. The percentage of regulation as measured by tvDTH assay in children with testable non-inherited maternal alleles. Male ( ) and female (D) children either had none (open symbols) or some (half filled and filled) late amplicons from NIMA alleles. The majority of individuals with late NIMA loci amplicons did not amplify at all loci (half filled). Red outlines indicate late amplicons of alleles not from mother.(male or somatic)
mismatched primer set. Using replicate reactions with different PCR chemistries, comparing reaction efficiencies, amplicon melt curves as well as molecular weight by gel electrophoresis certainly improves our ability to eliminate false positives. Unfortunately, it is still possible that unknown heterozygosity, higher rates of somatic mutation, template damage during purification, as well as mutation during synthesis in or near the critical mismatched primer binding site could contribute to a slightly more favorable late cycle binding and extension of an amplicon. Finally simply using highly complex template like genomic DNA provides a significant variation and number of primer binding sites that variably alter the primers in the reaction and impact the detection of true positive target amplicons. The purification of many templates that did not result in amplification after 50 cycles of each of the 2 InDel alleles supports the interpretation that the majority of correctly sized amplicons repeatedly detected after many PCR cycles in multiple aliquots from specific individuals are true positives. However there are trends that indicate further validation may be warranted. For example 6 out of 8 of the InDel loci show a distinctly higher frequency of late amplification of deletion alleles than insertion alleles and in particular S08 and S09 both have very high frequencies of late cycle amplifying deletion alleles when templates appear to generally lack those alleles. This could indicate either a rare higher somatic mutation rate for the
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occurrence of deletion alleles in individuals homozygous for the insertion or that some primer pairs may be more or less sensitive to late cycle reaction conditions giving rise to false positives. It is possible that this somatic mutation rate is attributable to one specific cell type (e.g., neutrophils), however preliminary comparisons of PBMC with PMN fractions did not show consistent significant differences in detecting the rare templates. Unfortunately this does not clearly resolve the question since typical gradient purified fractions contain about 10% contamination of the other fraction (PMN in PBMC and visa versa). A related issue is whether the short-lived rapid growth of the neutrophils might create a distorted sampling that somehow overlooks or over-emphasizes a particular clonal microchimerism. This is why we thoroughly tested male microchimerism. We feel that the frequency of male microchimerism detected in our study is similar to several previous reports and supports the notion that we are detecting similar levels of microchimerism.16,28,29,31 In a recent investigation of native hematopoiesis in mice, single cell PCR of 290 neutrophils indicated 270 were derived from uniquely tagged stem cell clones without indication of over-representation of any particular clone.34 This extreme clonal diversity of the PMN fraction could reduce the level of template below the level of detection. However, the consistent repeatability in our study of both male and insertion/deletion loci suggests we could consistently detect microchimerism even in this highly polyclonal population. In native hematopoiesis in mice, many lymphocyte (T and B cell) clones were not shared with the PMN (granulocyte) cells.34 This could partially explain the distinct levels of microchimerism that have been reported when looking at specific longer-lived cell-types.16,35 In a primate study of recovering hematopoiesis, there were not a large number of clones specific for the peripheral T and B cells.36 Instead, NK cells were found to be not only lineage specific clones but at several sampling times had overrepresentation of particular clones.36 Distorted sampling like this might make it easier or harder to detect microchimerism with any given sample, however, this type of bias was seen in the PMN cells only very early during the recovery of hematopoiesis.36 Another concern is that while many individuals had multiple testable NIMA alleles, many of these individuals did not test positive for all of the potential NIMA alleles. This could reflect variation in the sensitivity of reactions caused by variation in the complex template from individual to individual. The efficiency of primers binding to alternative sites could prevent those primers from amplifying the true rare target in a particular genetic background. If these are produced efficiently they can competitively exclude unaltered primers from binding the true target. For example in some cases individual templates had variation in sequences that allowed for the efficient amplification of competing amplicons with unique but incorrect molecular weight (unpubl. observations). These alternate products will not only rapidly remove primers from the pool of primers needed to amplify rare templates but their presence could interfere with the unaltered primer binding to the true target during late cycles of the PCR. If the majority of the late amplifying potential NIMA alleles actually are reporting rare maternal cells then the data clearly indicates that some individuals have regulation without detectable peripheral PMN fraction microchimerism. This may simply be due to this fraction not always representing maternal cells
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hidden in the bone marrow, organs and tissues that maintain a selection for the regulatory cells detected by tvDTH. On the other hand, the maintenance of microchimeric cells in individuals who do not have a regulated response seems to indicate that tolerance to the cells involves other mechanisms. Early publications from our lab showed evidence that microchimerism maybe associated with an induction of clonal anergy.37 It has also been reported that donor microchimerism can influence the deletion of donor specific CD8+ effector T cells.38 If microchimerism has been established in the thymus the routine deletion of NIMAspecific CD8+ effector cells might eliminate the need to maintain a regulated response. Many of the tvDTH assays reported here included lysate only injections that often showed no clear strong NIMA specific effector response. While additional speculation could be attempted the most significant issue with the data presented is that neither late cycle amplicons nor tvDTH may provide strong irrefutable evidence for long-term retention of maternal cells. Theoretically the best experimental design would be to isolate human stem cells from bone marrow, differentiate them and look for cells expressing NIMA specific antigens (e.g. HLA) by flow sorting then interrogate by next generation sequencing. A report that describes cells with hundreds of alleles from mother would provide irrefutable evidence for the long-term retention in human adults of maternal cells. Conclusion We failed to find a correlation between maternal microchimerism (MMc) in the PMN fraction of PBMC on the one hand, and immune regulation toward maternal antigens on the other hand. This finding is in agreement with previous failed attempts to find a correlation between HY antigen-specific immune regulation and SRY microchimerism in various PBMC subsets in women. Our findings differ from that of mouse studies in which all types of tissue, including bone marrow, lymphoid, and non-lymphoid organs were sampled, and correlation between Treg activity and MMc level was found. Accurate comparison of rare antigen source (microchimerism) with antigen-specific Treg response in humans may therefore require an approach that is not entirely PBMC-dependent, but samples other tissue sites as well. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
The authors would like to acknowledge Alex Blitman for his assistance in processing PMN fractions and the dozens of gels he helped to run. In addition, the authors acknowledge the contribution of the remaining undergraduate students (Sam Katers, Ross Ryan, Matt Pestrak and John Kernian) the graduate students (Vrushali Agashe, Mathew Brown, and William Bracamonte-Baran) the visiting surgeons (Miwa Satomi and Yusuke Tomita) and other staff members of the lab (Ying Zhou, and Jeremy Sullivan) for their daily suggestions, reviews and comments during the investigation.
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References 1. Lopez MM, Valenzuela JE, Alvarez FC, Lopez-Alvarez MR, Cecilia GS, Paricio PP. Long-term problems related to immunosuppression. Transplant Immunol 2006; 17:31-5 2. Jolly EC, Watson CJE. Modern immunosuppression. Surgery 2011; 29:312-8 3. Owen RD. Immunogenetic Consequences of Vascular Anastomoses between Bovine Twins. Science 1945; 102:400-1; PMID:17755278 4. Owen RD, Wood HR, Foord AG, Sturgeon P, Baldwin LG. Evidence For Actively Acquired Tolerance To Rh Antigens. Proc Natl Acad Sci USA 1954; 40:420-4; PMID:16589498 5. Maloney S, Smith A, Furst DE, Myerson D, Rupert K, Evans PC, Nelson JL. Microchimerism of maternal origin persists into adult life. J Clin Invest 1999; 104:417; PMID:10393697 6. Burlingham WJ, Grailer AP, Heisey DM, Claas FH, Norman D, Mohanakumar T, Brennan DC, de Fijter H, van Gelder T, Pirsch JD, et al. The effect of tolerance to noninherited maternal HLA antigens on the survival of renal transplants from sibling donors. New Eng J Med 1998; 339:1657-64; PMID:9834302 7. Andrassy J, Kusaka S, Jankowska-Gan E, Torrealba JR, Haynes LD, Marthaler BR, Tam RC, Illigens BM, Anosova N, Benichou G, et al. Tolerance to noninherited maternal MHC antigens in mice. J Immunol 2003; 171:5554-61 8. Molitor-Dart ML, Andrassy J, Kwun J, Kayaoglu HA, Roenneburg DA, Haynes LD, Torrealba JR, Bobadilla JL, Sollinger HW, Knechtle SJ, et al. Developmental exposure to noninherited maternal antigens induces CD4+ T regulatory cells: relevance to mechanism of heart allograft tolerance. J Immunol (Baltimore, Md : 1950) 2007; 179:6749-61 9. Molitor-Dart ML, Andrassy J, Haynes LD, Burlingham WJ. Tolerance induction or sensitization in mice exposed to noninherited maternal antigens (NIMA). Am J Transplant 2008; 8:2307-15; PMID:18925902 10. Dutta P, Burlingham WJ. Stem cell microchimerism and tolerance to non-inherited maternal antigens. Chimerism 2010; 1:2-10; PMID:21132055 11. Dutta P, Molitor-Dart M, Bobadilla JL, Roenneburg DA, Yan Z, Torrealba JR, Burlingham WJ. Microchimerism is strongly correlated with tolerance to noninherited maternal antigens in mice. Blood 2009; 114:3578-87; PMID:19700665 12. Akiyama Y, Caucheteux Sm, Vernochet C, Vernochet C, Iwamoto Y, Tanaka K, Kanellopoulos-Langevin C, Benichou G. Transplantation tolerance to a single noninherited MHC class I maternal alloantigen studied in a TCR-transgenic mouse model. J Immunol 2011; 186 (3-4):1442-9 13. Kinder JM, Jiang TT, Ertelt JM, Xin L, Strong BS, Shaaban AF, Way SS. Cross-Generational Reproductive Fitness Enforced by Microchimeric Maternal Cells. Cell 2015; 162:505-15; PMID:26213383 14. VanBuskirk AM, Burlingham WJ, Jankowska-Gan E, Chin T, Kusaka S, Geissler F, Pelletier RP, Orosz CG. Human allograft acceptance is associated with immune regulation. J Clin Invest 2000; 106:145-55; PMID:10880058
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5:359-63; PMID:1791857; http://dx.doi.org/10.1016/ S0890-8508(06)80007-1 Johnson KL, Dukes KA, Vidaver J, LeShane ES, Ramirez I, Weber WD, Bischoff FZ, Hahn S, Sharma A, Dang DX, et al. Interlaboratory comparison of fetal male DNA detection from common maternal plasma samples by real-time PCR. Clin Chem 2004; 50:51621; PMID:14718394; http://dx.doi.org/10.1373/ clinchem.2003.024380 Reynolds N, Cooke HJ. Role of the DAZ genes in male fertility. Reprod Biomed Online 2005; 10:72-80; PMID:15705297; http://dx.doi.org/10.1016/S14726483(10)60806-1 Lau YF, Li Y, Kido T. Gonadoblastoma locus and the TSPY gene on the human Y chromosome. Birth Defects Res Part C, Embryo Today 2009; 87:114-22; http://dx.doi.org/10.1002/bdrc.20144 Lambert NC, Lo YM, Erickson TD, Tylee TS, Guthrie KA, Furst DE, Nelson JL. Male microchimerism in healthy women and women with scleroderma: cells or circulating DNA? A quantitative answer. Blood 2002; 100:2845-51; PMID:12351394; http://dx.doi.org/ 10.1182/blood-2002-01-0295 Bianchi DW, Farina A, Weber W, Delli-Bovi LC, Deriso M, Williams JM, Klinger KW. Significant fetalmaternal hemorrhage after termination of pregnancy: implications for development of fetal cell microchimerism. Am J Obstet Gynecol 2001; 184:703-6; PMID:11262475; http://dx.doi.org/ 10.1067/mob.2001.111072 Wataganara T, Chen AY, LeShane ES, Sullivan LM, Borgatta L, Bianchi DW, Johnson KL. Cell-free fetal DNA levels in maternal plasma after elective first-trimester termination of pregnancy. Fertil Steril 2004; 81:638-44; PMID:15037414; http://dx.doi.org/ 10.1016/j.fertnstert.2003.07.028 Sun J, Ramos A, Chapman B, Johnnidis JB, Le L, Ho YJ, Klein A, Hofmann O, Camargo FD. Clonal dynamics of native haematopoiesis. Nature 2014; 514:322-7; PMID:25296256; http://dx.doi.org/ 10.1038/nature13824 Loubiere LS, Lambert NC, Flinn LJ, Erickson TD, Yan Z, Guthrie KA, Vickers KT, Nelson JL. Maternal microchimerism in healthy adults in lymphocytes, monocyte/macrophages and NK cells. Lab Invest 2006; 86:1185-92 Wu C, Li B, Lu R, Koelle SJ, Yang Y, Jares A, Krouse AE, Metzger M, Liang F, Lore K, et al. Clonal tracking of rhesus macaque hematopoiesis highlights a distinct lineage origin for natural killer cells. Cell Stem Cell 2014; 14:486-99; PMID:24702997; http://dx.doi.org/ 10.1016/j.stem.2014.01.020 Burlingham WJ, Grailer AP, Fechner JH, Jr., Kusaka S, Trucco M, Kocova M, Belzer FO, Sollinger HW. Microchimerism linked to cytotoxic T lymphocyte functional unresponsiveness (clonal anergy) in a tolerant renal transplant recipient. Transplantation 1995; 59:1147-55; PMID:7732562; http://dx.doi.org/ 10.1097/00007890-199504270-00013 Bonilla WV, Geuking MB, Aichele P, Ludewig B, Hengartner H, Zinkernagel RM. Microchimerism maintains deletion of the donor cell-specific CD8+ T cell repertoire. J Clin Invest 2006; 116:156-62; PMID:16395404; http://dx.doi. org/10.1172/JCI26565
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Microchimerism and regulation in living related kidney transplant families W John Haynes, Ewa Jankowska-Gan, Lynn Haynes, and William J Burlingham* Department of Surgery; School of Medicine and Public Health; University of Wisconsin; Madison, WI USA
Long-term harmful effects of immunosuppressive drugs and chronic rejection are a persistent impetus to establish methods to induce immunological tolerance to allografts. PCR-based studies have found evidence that humans and other placental mammals can have prolonged extremely low levels of maternal cells as well as other non-self cells, referred to as microchimerism. The persistence of these cells suggests a mechanism for the maintenance of the regulatory T-cell (Treg) responses frequently detected in offspring to non-inherited maternal antigens. We test the hypothesis that the detection of very low copy levels of insertion/deletion (Indel) alleles consistent with non-inherited maternal genes, will correlate with immune regulation to non-inherited maternal antigens as detected by a trans-vivo Delayed-Type Hypersensitivity (tvDTH) assay in kidney transplant recipients, normal donors and their immediate biological family members. Preliminary data reported here compares qPCR amplification of rare DNA templates in the peripheral blood polymorphonuclear (PMN) fraction of cells, with the results of tvDTH assays for linked suppression of recall antigen responses in the presence of non-inherited maternal antigens [NIMA]. The two assays do not show a definitive correlation.
Introduction The goal of transplanted organs providing a cure for a variety of diseases is limited by the long-term chronic toxicity of the immunosuppressive drugs currently required to regulate the immune response to non-self antigens and maintain good graft function.1 Efforts to reduce the toxicity have led to new drugs and different maintenance therapies.2 However the hope of eliminating the need for these drugs requires expanding our understanding of how several mechanisms (anergy, clonal deletion and regulation) establish tolerance to non-self antigens. The earliest publication of natural tolerance in mammals was the description of freemartin cattle by Ray Owen in 1945.3 Owen later reported the first evidence for acquired tolerance to non-inherited maternal antigens (NIMA) in humans.4 Since then numerous studies have attempted to characterize the details of the mechanism(s) involved in the tolerance to NIMA.5-10 In mice, backcrossing a maternal hybrid [C57BL/6 (H2b) x DBA/2 (H2d)] that is heterozygous at the MHC locus (i.e. H2) with a homozygous (b\b) male resulted in half of the (b\b) offspring able to accept a fully allogeneic heart (d\d) without immunosuppresion.7 This capacity was strongly correlated with T reg activity detected pre-transplant and long-term persistence of a maternal allele indirectly measured by late cycle PCR.11 The persistence of the maternal microchimerism was curiously dependent on exposure to the maternal antigens during nursing and maybe related to the maturation of the mouse immune system after birth.11 The proposed theory was that maintenance of the NIMA-specific TGFbC CD4+ Treg cells required continuous
exposure to the antigen provided by persistent maternal microchimerism in several organs as well as in the bone marrow.8,12 Recent intricate experiments have provided striking evidence that an adult female, exposed in utero to a transgenic NIMA, when treated with an antibody against the same transgenic NIMA rapidly loose Treg cells specific for the NIMA construct.13 The inference is that elimination of cells expressing the antigen negatively impacts the maintenance of Treg to those antigens. More interestingly, in adult females there was a dramatic impact on the ability to maintain a pregnancy when the Tregs to antigens expressed by the fetus were eliminated. In humans, the trans-vivo delayed-type hypersensitivity assay (tvDTH) is one of the very few assays that measures regulation to specific antigens and has continued to provide evidence for NIMA regulation.14-16 The assay places cells from humans into the footpad of a severe combined immunodeficient (SCID) mouse along with a known potent recall antigen (e.g. tetanus). This elicits a swelling response dependent on the activation of established memory-effector T-cells by way of appropriate antigen presenting cells in the injected cell population. In specific cases, the injection of additional secondary antigens decreases (or rapidly resolves) the swelling response.15 This reduced swelling is referred to as linked suppression (or regulation) and requires both dendritic cells and antigen-specific T regulatory cells. This assay has revealed that cells from the peripheral blood of offspring very frequently show a regulated response to non-inherited maternal antigens in humans [the NIMA effect17] The fact that large prospective studies of outcomes among human living related kidney transplant populations do not always show a clear
*Correspondence to: William J Burlingham; Email: [email protected] Submitted: 06/25/2015; Revised: 09/15/2015; Accepted: 10/16/2015 http://dx.doi.org/10.1080/19381956.2015.1111974
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beneficial effect of maternal over paternal mismatched kidney transplants (NIMA vs. NIPA), demonstrate that other factors must be involved.18,19 One such factor is the response of the donor to the recipient. It has been shown that between mothers and their offspring, a mutual or bi-directional regulation appears to be of clear benefit in living related kidney transplants.17,20 In this report we present data to test the hypothesis that there is a correlation between regulation to non-inherited maternal antigens among family members and evidence for persistent microchimeric maternal alleles indirectly detected by PCR. The tvDTH assay requires the separation of peripheral blood mononuclear cells (PBMC) from a very abundant polymorphonuclear (PMN) cell fraction. The significant numbers of PBMC used in the tvDTH assay often limited the number of cells that could be routinely used to extract total DNA template for qPCR, whereas the PMN fraction was often discarded. The PMN fraction typically also contains about 10% lymphocytes, and therefor is a readily available, abundant pool of peripheral blood cells that could be routinely used to extract DNA. The majority of the haematopoietic cells in the bone marrow develop into myeloid cells and specifically into mature neutrophils. This population of cells has a very short half-life in the peripheral blood (8-12 hours to perhaps up to 5 days or longer),21 the cells continuously egress from the bone marrow and a previous publication has reported detecting maternal microchimerism in this cell fraction.22 These cells therefor should reflect a continuously renewed sampling of haematopoietic precursor cells from the bone marrow and provide indirect evidence for maternal cells in the bone marrow.
Material and Methods Study participants Immunological monitoring was performed on samples obtained from healthy family members and end stage renal disease (ESRD) patients according to informed consent procedures, subject to human subjects Institutional Review Board approval at the University of Wisconsin-Madison. Isolation of leukocyte cell types Peripheral blood mononuclear cells (PBMC) used in the tvDTH were the entire mixed “buffy coat” of cells (T-cells, B-cells, monocytes, and a significant frequency of contaminating neutrophils) isolated by density gradient centrifugation using a sterile, iso-osmotic polysucrose and diatrizoate solution (Lymphocyte Separation Medium; Corning). Cells for chimerism testing were obtained from the remaining fraction of cells after lysing red blood cells with NH4Cl and KHCO3 lysis (ACK) buffer. The majority of the cells in this fraction were polymorphonuclear (PMN; a.k.a. neutrophils) with an average of about 10% mononuclear lymphocytes. Testing Treg function in the trans vivo Delayed Type Hypersensitivity assay Fresh human PBMC [7–9£106] in PBS were mixed with cell sonicate-derived antigen prepared from PBMC and injected into the footpad of 6-8 week old CB.17 SCID mice, as described
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previously.14,15,23 The response to tetanus toxoid recall antigen alone plus PBMC was used as a positive control, with PBMC alone as a negative control. To test for bystander suppression, a recall antigen was co-injected with test antigen (antigen from family members). Footpad thickness was measured using a spring-loaded caliper at time 0 (before injection) and 24 hours after injection. Net swelling was determined by subtracting “background” swelling from a control injection of PBMC alone. The extent of bystander suppression was measured as % inhibition of the recall antigen response in the presence of test antigen, calculated using the following formula: % Inhibition (or % Regulation) D [1-(recall+test antigen)/ recall alone] x 100% Total DNA isolation Each sample processed contained 2£107 cells from the PMN fraction from the same peripheral blood samples used in the tvDTH assays. Purification was done using QIAmp DNA blood minikit (QIAGEN) with minor modifications. An RNase cocktail (Ambion) was used to insure the removal of all RNA from the sample. During the 56 C incubation used to digest protein and RNA, a syringe was used to disperse cellular material to facilitate digestion. After addition of Ethanol, repeated pipetting along the side of the tube was done to facilitate fragmenting DNA. Experiments determined that column purification does not work efficiently for fragments larger than 25 Kb and fragments 50 Kb or larger do not elute from the columns (data not shown). The dispersion during digestion and the fragmentation by pipetting increased the average yield 2-4 fold. Total nucleic acid concentration of each sample was quantified on a NanoDrop 1000 spectrophotometer (Thermo Scientific). Genomic DNA fragments in samples were examined using intercalating Ethidium Bromide after separation by 0.7% agarose gel electrophoresis to ensure fragmentation was minimal and all RNA had been removed. All PCR products were also examined and validated by 2.0% agarose gel electrophoresis. Detection of microchimeric template variation A set of previously published primers and probes were used to distinguish insertion deletion alleles that maintain a high degree of heterozygosity at 8 somatic loci and one X-linked locus in humans.24,25 In addition, primers and probes for 3 loci on the male chromosome were used to test if the frequency of male microchimerism was similar to previous reports.26-29 The DAZ (DYS1) primers and TSPY (DYS14) primers amplify 2 different polyploid sequences on the Y chromosome. The actual copy number appears to be somewhat variable.29,30 The TSPY primers and probe used here are newly designed shifting the amplicon (a PCR amplified product) 75 bases from the original DYS14 primers first used by Lo et al. and later by Lambert et al.26,31 (sequence available upon request). The DAZ primers were the same as previously described.32,33 The SRY primers amplify a single copy locus and along with the GAPDH positive control were described by Johnson et al.28 Both SYBR green (SABioscience and later Qiagen) and probe-based (SsoFast; BioRad) qPCR chemistry were used in several replicate reactions. Patients
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and family members were screened to determine the alleles inherited at a normal complement level. The screen was generally done at a minimum of 2 concentrations of template (neet and diluted to either 20, 50 or 100ng/ml). Template was also tested after digestion with the restriction enzyme HinDIII, known not to cut any of the amplicons, to ensure maximum amplification efficiency was tested. Efforts to use high concentrations (>20 ng/ ml of reaction) proved to inhibit amplification and frequently reduced the sensitivity of the assays. The condition used in general for the screen reported here were multiple 10ml reactions held at 95 C for 5 minutes, then cycled from 95 C for 5 seconds to 60 or 62 C annealing and extension for 15 seconds in a quick 2 step PCR for 50 cycles. Dilutions of positive template into negative template demonstrated that the assays were generally sensitive enough to amplify as little as a few and sometimes as little as one copy of target loci per tube. Results QPCR analysis of microchimerism The cohort reported here includes 16 mothers, 4 fathers, and 32 offspring for a total of 52 individuals examined by qPCR at 12 loci, including 15 ESRD patients and their paired normal donors (Table 1). The male loci tested have varying degrees of natural ploidy along the Y chromosome and were used to assess whether detection of male microchimerism correlated with the expected copy number variation and other published data. The results of the screen for male microchimerism found 41% and 38% late cycle amplicons in the 2 polyploid loci (DAZ, TSPY) and 21% at the single copy locus (SRY; Table 1). The efficiency of amplification of the 2 different polyploid loci amplicons did not result in significant difference in the detection of male microchimerism. The overall frequency of male microchimerism found is similar to the frequency previously reported.16,31 The insertion/deletion (InDel) loci heterozygosity in this cohort of 52 individuals was between 43 and 67% (Table 1). Control reactions without template typically yielded no products
except for infrequent primer dimers or accidental contamination from a neighboring well. These events were extremely low frequency, had very late amplification with distinctly different amplification efficiencies. Templates from many individuals that did not inherit one of the alleles routinely had no amplification or infrequently distinctly incorrect alternate products, again with different amplification efficiencies and amplicon lengths. Every reaction was examined by both gel electrophoresis and second derivative melting curves. In contrast, templates from some individuals routinely generated distinctly late amplifying products of the expected size (Fig. 1A). These low copy alleles on average appeared with a ΔCt of between 10 and 15 compared with both the alternate allele and GAPDH as normal genetic complement controls. The late cycle amplicons, generally, amplified earlier than other types of false positive (primer dimers, etc.) and had amplification efficiencies similar to true positive amplicons (Fig. 1B). Each negative allele showing signs of late amplifying product were re-examined by replicate qPCR in comparison with template that was a consistent negative for the allele and a second template with consistent positive normal complement allele. This particular example is an ESRD patient and the living related donor. The ESRD patients and normal living donors showed no significant difference in the frequency of late cycle (low copy) amplification of products (ND15; 98 and 104 low copy or negative alleles tested, respectively). Screening this population for testable NIMA loci, a total of 23 individual offspring were available to examine for potential noninherited maternal late cycle amplicons and correlate with the regulation assay (Fig. 2). 5 females (D) and 6 males ( ) show no evidence of late amplifying non-inherited maternal amplicons (No NIMA Mc). 4 individuals in this group had late amplification of alleles that were not NIMA (red outline; either microchimerism in alleles not carried by mother or male microchimerism). In the second group there were 12 individuals that had at least one allele with a late cycle NIMA amplicon, and some individuals also had late cycle nonNIMA amplicons. There was clearly no statistically significant difference between these 2 groups with respect to regulation using either unpaired T-test or a Mann-Whitney non-parametric test (p>0.5).
Table 1. Summary statistics of the PCR for 52 individuals. 18 males and 34 women (Median age: 54; minimum age: 21). 16 of the women had late cycle male amplicons at either the polyploid loci DAZ, or TSPY, and in some cases the single locus SRY (Row 3, first 3 columns). The polyploid loci were consistently detected at an earlier cycle than the single locus SRY, but in some women only the polyploid loci were amplified. The remaining somatic InDel loci were generally of high heterozygosity (Row 7) and could be frequently interrogated for low copy templates (row 8) and despite differences in reaction kinetics showed a similar average ΔCq with the reference GAPDH (row 9). Male Loci DAZ TSPY SRY 1) positive 2) negative 3) late cycle amplicon 4) Total negative+late 5) Total defined alleles 6) % negative or late 7) Heterozyogousity 8) % of negative w/late amplicon 9) Average DCq (Loci-GAPDH)
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S01 del
S04 in
del
18 18 18 39 42 37 18 20 27 12 9 9 16 14 7 1 1 6 34 34 34 13 10 15 52 52 52 52 52 52 65% 65% 65% 25% 19% 29% — — — 44.2% 42.3% 47% 41% 21% 8% 10% 40% 14 13 18 12 15 14
S07 in S06
del
45 7 0 7 52 13%
28 40 20 9 4 3 24 12 52 52 46% 23% 67.3% 0% 17% 25% — 16 15
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S08 in
del
29 44 18 2 5 6 23 8 52 52 44% 15% 42.3% 22% 75% 15 14
S09 in
38 8 6 14 52 27%
del
49 1 2 3 52 6% 65.4% 43% 67% 13 15
S10 in
del
21 40 26 9 5 3 31 12 52 52 60% 23% 46.2% 16% 25% 14 13
S11 in
del
Tg in
38 33 12 14 2 5 14 19 52 52 27% 37% 46.2% 14% 26% 19 15
del
47 31 5 16 0 5 5 21 52 52 10% 40% 57.7% 0% 24% — 16
in 43 8 1 9 52 17% 11% —
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Figure 1. Example of a late cycle amplification. (A) Early amplification of a heterozygous template (donor; 298.2 ng/10ml; gel lane 2) for both S08 insertion and deletion alleles compared with a template homozygous for S08 insertion (recipient; 292 ng/10 ml; gel lane 4) with a late amplifying S08 deletion (recipient; gel lane 3) amplicon. A positive GAPDH control for both templates (gel lane 5). (B) Overlapping derivative melt curves for 3 products S08 insertion, S08 deletion and GAPDH show slight shifts in peak melting temperature.
Discussion The data indicates no clear relationship between the late cycle amplification products from the PMN fraction and regulation to NIMA as measured by the tvDTH assay. This is a very similar result to one recently published looking for a correlation of male microchimerism with regulation to minor HY antigens in a variety of cell fractions emphasizing different cell types.16 In this report, the clear and repeatable detection of late cycle amplicons in specific templates substantiates the usefulness of the InDel primers and the PMN fraction in looking for alternate low copy alleles. The PMN fraction provides a very large numbers of cells that can be examined while leaving the PBMC for tvDTH or other regulation assays. In addition, the tendency for these cells to maintain more diffuse, less compact nuclei allows DNA to be more efficiently purified and ready for PCR amplification (personal observation). The challenge in making strong conclusions from the PCR data presented here is that there are always improbable but potential reasons for late cycle amplification from the expected locus with a
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Figure 2. The percentage of regulation as measured by tvDTH assay in children with testable non-inherited maternal alleles. Male ( ) and female (D) children either had none (open symbols) or some (half filled and filled) late amplicons from NIMA alleles. The majority of individuals with late NIMA loci amplicons did not amplify at all loci (half filled). Red outlines indicate late amplicons of alleles not from mother.(male or somatic)
mismatched primer set. Using replicate reactions with different PCR chemistries, comparing reaction efficiencies, amplicon melt curves as well as molecular weight by gel electrophoresis certainly improves our ability to eliminate false positives. Unfortunately, it is still possible that unknown heterozygosity, higher rates of somatic mutation, template damage during purification, as well as mutation during synthesis in or near the critical mismatched primer binding site could contribute to a slightly more favorable late cycle binding and extension of an amplicon. Finally simply using highly complex template like genomic DNA provides a significant variation and number of primer binding sites that variably alter the primers in the reaction and impact the detection of true positive target amplicons. The purification of many templates that did not result in amplification after 50 cycles of each of the 2 InDel alleles supports the interpretation that the majority of correctly sized amplicons repeatedly detected after many PCR cycles in multiple aliquots from specific individuals are true positives. However there are trends that indicate further validation may be warranted. For example 6 out of 8 of the InDel loci show a distinctly higher frequency of late amplification of deletion alleles than insertion alleles and in particular S08 and S09 both have very high frequencies of late cycle amplifying deletion alleles when templates appear to generally lack those alleles. This could indicate either a rare higher somatic mutation rate for the
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occurrence of deletion alleles in individuals homozygous for the insertion or that some primer pairs may be more or less sensitive to late cycle reaction conditions giving rise to false positives. It is possible that this somatic mutation rate is attributable to one specific cell type (e.g., neutrophils), however preliminary comparisons of PBMC with PMN fractions did not show consistent significant differences in detecting the rare templates. Unfortunately this does not clearly resolve the question since typical gradient purified fractions contain about 10% contamination of the other fraction (PMN in PBMC and visa versa). A related issue is whether the short-lived rapid growth of the neutrophils might create a distorted sampling that somehow overlooks or over-emphasizes a particular clonal microchimerism. This is why we thoroughly tested male microchimerism. We feel that the frequency of male microchimerism detected in our study is similar to several previous reports and supports the notion that we are detecting similar levels of microchimerism.16,28,29,31 In a recent investigation of native hematopoiesis in mice, single cell PCR of 290 neutrophils indicated 270 were derived from uniquely tagged stem cell clones without indication of over-representation of any particular clone.34 This extreme clonal diversity of the PMN fraction could reduce the level of template below the level of detection. However, the consistent repeatability in our study of both male and insertion/deletion loci suggests we could consistently detect microchimerism even in this highly polyclonal population. In native hematopoiesis in mice, many lymphocyte (T and B cell) clones were not shared with the PMN (granulocyte) cells.34 This could partially explain the distinct levels of microchimerism that have been reported when looking at specific longer-lived cell-types.16,35 In a primate study of recovering hematopoiesis, there were not a large number of clones specific for the peripheral T and B cells.36 Instead, NK cells were found to be not only lineage specific clones but at several sampling times had overrepresentation of particular clones.36 Distorted sampling like this might make it easier or harder to detect microchimerism with any given sample, however, this type of bias was seen in the PMN cells only very early during the recovery of hematopoiesis.36 Another concern is that while many individuals had multiple testable NIMA alleles, many of these individuals did not test positive for all of the potential NIMA alleles. This could reflect variation in the sensitivity of reactions caused by variation in the complex template from individual to individual. The efficiency of primers binding to alternative sites could prevent those primers from amplifying the true rare target in a particular genetic background. If these are produced efficiently they can competitively exclude unaltered primers from binding the true target. For example in some cases individual templates had variation in sequences that allowed for the efficient amplification of competing amplicons with unique but incorrect molecular weight (unpubl. observations). These alternate products will not only rapidly remove primers from the pool of primers needed to amplify rare templates but their presence could interfere with the unaltered primer binding to the true target during late cycles of the PCR. If the majority of the late amplifying potential NIMA alleles actually are reporting rare maternal cells then the data clearly indicates that some individuals have regulation without detectable peripheral PMN fraction microchimerism. This may simply be due to this fraction not always representing maternal cells
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hidden in the bone marrow, organs and tissues that maintain a selection for the regulatory cells detected by tvDTH. On the other hand, the maintenance of microchimeric cells in individuals who do not have a regulated response seems to indicate that tolerance to the cells involves other mechanisms. Early publications from our lab showed evidence that microchimerism maybe associated with an induction of clonal anergy.37 It has also been reported that donor microchimerism can influence the deletion of donor specific CD8+ effector T cells.38 If microchimerism has been established in the thymus the routine deletion of NIMAspecific CD8+ effector cells might eliminate the need to maintain a regulated response. Many of the tvDTH assays reported here included lysate only injections that often showed no clear strong NIMA specific effector response. While additional speculation could be attempted the most significant issue with the data presented is that neither late cycle amplicons nor tvDTH may provide strong irrefutable evidence for long-term retention of maternal cells. Theoretically the best experimental design would be to isolate human stem cells from bone marrow, differentiate them and look for cells expressing NIMA specific antigens (e.g. HLA) by flow sorting then interrogate by next generation sequencing. A report that describes cells with hundreds of alleles from mother would provide irrefutable evidence for the long-term retention in human adults of maternal cells. Conclusion We failed to find a correlation between maternal microchimerism (MMc) in the PMN fraction of PBMC on the one hand, and immune regulation toward maternal antigens on the other hand. This finding is in agreement with previous failed attempts to find a correlation between HY antigen-specific immune regulation and SRY microchimerism in various PBMC subsets in women. Our findings differ from that of mouse studies in which all types of tissue, including bone marrow, lymphoid, and non-lymphoid organs were sampled, and correlation between Treg activity and MMc level was found. Accurate comparison of rare antigen source (microchimerism) with antigen-specific Treg response in humans may therefore require an approach that is not entirely PBMC-dependent, but samples other tissue sites as well. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
The authors would like to acknowledge Alex Blitman for his assistance in processing PMN fractions and the dozens of gels he helped to run. In addition, the authors acknowledge the contribution of the remaining undergraduate students (Sam Katers, Ross Ryan, Matt Pestrak and John Kernian) the graduate students (Vrushali Agashe, Mathew Brown, and William Bracamonte-Baran) the visiting surgeons (Miwa Satomi and Yusuke Tomita) and other staff members of the lab (Ying Zhou, and Jeremy Sullivan) for their daily suggestions, reviews and comments during the investigation.
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