more than 50 years ago , owen et al . ( 1954 ) made the remarkable discovery that most twin cattle were born with a stable mixture of each other s red cells .
( 1988 ) later found that human leukocyte antigen ( hla ) broadly sensitized patients commonly failed to produce antibodies against mismatched non - inherited maternal antigens ( nima ) , but were fully capable of producing anti - non - inherited paternal antigens ( nipa ) .
the definition of nima or nipa is based on an offspring - based hla haplotype that is not inherited from the mother or father , respectively .
( 1953 ) then showed that injection of allogeneic splenocytes from murine fetuses enabled the acceptance of later skin grafts from the same donor .
this phenomenon is now referred to as fetomaternal tolerance , and suggests that perinatal exposure to nima may affect the developing immune system of neonates .
these phenomena have been clinically utilized in organ transplantation and allogeneic hematopoietic stem cell transplantation ( hsct ; burlingham et al . , 1998 ; van rood et al . , 2002 ) .
( 1998 ) showed the superior graft survival rate in nima- compared to nipa - mismatched renal transplant recipients from sibling donors .
furthermore , van rood et al . ( 2002 ) demonstrated that hsct from nima - mismatched sibling donors showed a lower incidence of severe acute graft - versus - host disease ( gvhd ) compared with that from the other family donors .
( 2004 ) have demonstrated the feasibility of hla - haploidentical hsct from nima - mismatched relatives without t cell depletion .
these clinical studies have been performed based on the presence of fetomaternal microchimerism as a result of fetomaternal immunological tolerance . nevertheless , some cases developed severe acute gvhd despite the existence of microchimeric cells ( kanda et al . , 2009 ) .
we recently reported that nima effects directed toward the major histocompatibility complex ( mhc ) antigen were divided into immunogenic and tolerogenic reactivities ( araki et al . , 2010 ) .
the reactivities were predictable by an mlr - elispot ( mixed lymphocyte reaction ; enzyme linked immunospot ) assay .
we found that non - t cell - depleted ( tcd ) nima - mismatched haploidentical hsct could be performed safely by evaluating the reaction of ifn--producing cells of the donors against nima before transplantation .
alloantigens can be divided into mhc antigen and minor histocompatibility antigen ( miha ) , the former being responsible for eliciting the strongest immune responses to allogeneic tissues .
the mhc is referred to as the hla complex in humans and as the h-2 complex in mice ( table 1 ) .
the genes related to the hla system encode a complex array of histocompatibility molecules that play a central role in immune responsiveness and in determining the outcome of hsct in humans ( beatty et al .
the primary goal of histocompatibility testing for patients who are undergoing hsct is the identification of a suitable hla - matched donor to reduce the risk of post - transplant complications , which may result from hla incompatibility .
the mhc identity of the donor and host is not the sole factor determining the immunological reactivity in hsct . when transplantation is performed in an unrelated setting , even if the mhc antigens of donor are identical to those of recipient , considerable transplant reactions may occur because of differences at various minor histocompatibility loci .
mihas , peptides derived from polymorphic proteins , are capable of eliciting cellular alloimmune responses in vitro and in vivo . their immunogenicity arises as a result of their presentation in the context of mhc class i or ii , where they are recognized by alloreactive mhc - restricted t cells .
the most important immune reactions elicited by in vivo alloreactivity to miha are graft rejection and acute gvhd . to date ,
human mihas have not been fully characterized , although some murine mihas have been compared with the human counterparts ( table 1 ) .
immunological targeting of hy proteins results in a relatively high incidence of acute gvhd when male recipients receive hsct from female donors ( stern et al . ,
while approximately one - third of the known mihas are encoded on the y chromosome , many mihas are located on autosomal chromosomes .
a genetic linkage analysis has been used to define the genomic regions encoding the mihas ( akatsuka et al . , 2003 ; de rijke et al . , 2005 ) .
with the recent introduction of more advanced analytical techniques , more human miha epitopes have been identified ( van bergen et al . , 2010 ; sellami et al . , 2011 )
graft survival in hsct is optimal when the donor and recipient are hla - identical .
however , in some situations , if this is not possible , haploidentical siblings , parents , and offspring are considered as potential donors .
contact between the mother and child during pregnancy can lead to tolerization , and subsequently have an additional benefit on the transplant outcome .
a new nomenclature was proposed to assign the haplotypes of a family in which one of the siblings is a potential transplant donor ( van rood and claas , 2000 ) as depicted in figure 1 .
the parents or siblings that share one haplotype with the recipient and differ for the other haplotype are potential donors .
the patient inherits the inherited maternal hla antigens ( i m a ) haplotype from the mother , and the inherited paternal hla antigens ( ipa ) from the father .
when the patient is transplanted from one of the parents or from a haploidentical sibling , the nima or nipa is the mismatched haplotype .
this nomenclature scheme can also be used in cases where the mother or father is the potential donor ( figure 1 ) . because of the existence of fetomaternal tolerance , nipa is more immunogenic than nima
. therefore , the order of donor eligibility is ima / ipa , nima / ipa followed by ima / nipa .
siblings of the patient share one haplotype with the donor , and the other haplotype is the non - inherited haplotype . the hla haplotypes in parentheses are shown as representative examples . when the patient is transplanted with donor from one of the parents or from a haploidentical sibling , the non - inherited maternal hla antigens ( nima ) or non - inherited paternal hla antigens ( nipa ) are the mismatched haplotype .
several studies have been performed to investigate the influence of non - inherited and inherited parental antigens on transplantation , and tolerizing effects ( a nima effect ) have been described . in hsct ,
van rood et al . ( 2002 ) and ichinohe et al . ( 2004 ) showed that the patients who received non - tcd bmt from a nima - mismatched donor had a significantly lower incidence of acute gvhd than a nipa - mismatched donor .
however , even in non - tcd bmt from a nima - mismatched donor , 10% of patients still experienced severe acute gvhd ( ichinohe et al . , 2004 ) .
furthermore , graft rejection and hyperacute gvhd after hsct from nima - mismatched siblings have been observed in spite of the fact that maternal microchimerism was detected ( okumura et al . , 2007 ) .
( 2009 ) described that a substantial proportion of long - term survivors after nima - mismatched hsct could discontinue the administration of immunosuppressive agents , despite the frequent occurrence of moderate to severe chronic gvhd .
there have been several investigations of nima in murine models ( burlingham et al . , 1998 ; andrassy et al . ,
the immunological effects of developmental exposure to nima are heterogeneous ( mold et al . , 2008 ; molitor - dart et al . , 2008 ; verhasselt et al . , 2008 ) .
not only in the mhc - identical , but also under mhc - haploidentical conditions , miha alloreactivities may be induced upon transplantation ( verdijk et al . , 2004 ) .
therefore , focusing on the nima effect separated by the mhc ( h-2 ) and miha responses is clinically relevant .
the mouse miha loci confer a wide range of immunogenicity , ranging from weakly to strongly immunogenic ( table 1 ; mendoza et al . , 1997 ; choi et al . , 2001
recent studies have provided evidence that gvhd could be caused by a limited number of miha , including h4 , h7 , h13 , h28 , h60 , and h - y ( eden et al .
, 1999 ; choi et al . , 2002 ; yang et al . , 2003 ) .
the immunodominance of these miha was manifested on genetically varied backgrounds among b10 , balb / c , and dba/2 strains ( sanderson and frost , 1974 ; mendoza et al . , 1997 ; malarkannan et al . , 2000
so far , there has been no report distinguishing h-2 from miha with regard to nima .
we have classified mouse models of nima based on the major and minor histocompatibility antigens to nima ( hirayama and azuma , 2011 ) . in our study
, b10 congenic mice were used as nima models and the miha matched entirely in this system ( figure 2b ) . on the other hand ,
in the conventional model ( figure 2a ) , the nima includes not only non - inherited h-2 , but also non - inherited miha .
therefore , our nima model , but not the conventional nima model , did not affect the immunogenicity of miha .
we examined the tolerogenic potential of nima - exposure for h-2 of class i and ii disparities without any influences of the miha ( araki et al . , 2010 ) .
2009 ) , we found no evidence of the nima effect ( araki et al . , 2010 ) .
the reason for the difference remains to be determined , but it could be due to the abrogation of the miha effect in our system ( figure 2b ) .
( a ) 
 left , c57bl/6 ( b6 ) males ( h-2 ) were mated with ( b6  dba/2 ) f1 females ( h-2 ) , thus exposing the h-2 offspring in utero and via breastfeeding to nima antigens .
right , ( b6  dba/2 ) f1 males were mated with b6 females , creating h-2 backcross offspring that had not been exposed to  d ,  as reported by andrassy et al .
( b ) 
 left , b10.br males ( h-2 ) were mated with ( b10.d2  b10 ) f1 females ( h-2 ) , thus exposing the h-2 type offspring to nima , and h-2 type offspring to nima .
right , ( b10.d2  b10 ) f1 males ( h-2 ) were mated with b10.br females ( h-2 ) , creating the controls ; both h-2 and h-2 type offspring that had not been exposed to  b  and  d ,  respectively .
these mice have a b10 background , in other words , their miha are matched , and the h-2 antigens are mismatched for both class i and ii .
the bidirectional exchange of cells , both mature and progenitor types , at the maternal  fetal interface is a common feature of mammalian reproduction ( lo et al . , 1996 ) .
the presence of semiallogeneic cells in a host can have significant immunological effects on transplantation tolerance and rejection .
maternal cells and dna were detected for a long time after parturition in the peripheral blood and lymphoid organs of offspring ( maloney et al .
breastfeeding during the neonatal period also might contribute to building - up maternal microchimerism in the offspring , because breast milk is rich in soluble maternal mhc antigens ( verhasselt et al .
maternal microchimerism may cause tolerance , resulting in the acceptance of an allograft bearing antigens shared by the microchimeric cells .
however , microchimerism may also cause sensitization , thus resulting in rejection . distinguishing which of these effects is likely to occur prior to the transplant may revolutionize the field of living - related renal transplantation , wherein microchimerism can exert a powerful influence on graft outcome ( van rood et al . ,
long - term maternal microchimerism is easily detected from the peripheral blood or various tissues , including the skin , liver , and thyroid gland , by using highly sensitive polymerase chain reaction - based techniques ( ichinohe et al . , 2002 ) .
although many investigators have suggested the association of long - term maternal and fetal microchimerism with the development of autoimmune diseases , including systemic sclerosis , primary biliary cirrhosis , juvenile inflammatory myopathies , and biliary atresia ( nelson , 2003 ; suskind et al . , 2004 ) , it is difficult to establish a precise etiological link , because maternal microchimerism is frequently found in healthy females with a history of uncomplicated pregnancy , and in more than two - thirds of immunocompetent individuals without any manifestations of autoimmune attacks ( kodera et al . , 2005 ) .
( 1999 ) suggested that passenger leukocytes were involved in the induction phase of allograft acceptance and microchimerism in the thymus , but not in the blood , and were also associated with allograft survival .
these results suggest that microchimerism may play a role in allograft survival , but that the persistence of peripheral microchimerism is not required .
( 2005 ) described partial deletion of b cells having high affinity for the nima .
( 1998 ) showed central and peripheral deletion of donor - specific t cells after establishing mixed chimerism in the recipient with a high dose of bone marrow cells .
low doses of bone marrow cells induced a form of tolerance that was regulatory t cells ( treg)-dependent , consistent with the establishment of microchimerism rather than mixed chimerism .
bonilla et al . ( 2006 ) described that deletion of effector t cells due to a form of suppressive microchimerism in antigen - presenting cells was a consequence of the establishment of a dominant treg - population in the host .
tsang et al . ( 2008 ) described the possibility of inducing nima - specific treg in the direct and indirect presentation of maternal microchimerism . on the other hand , since oral tolerance is known to generate tgf--producing treg ( gonnella et al . , 2003 ) , oral exposure to maternal mhc antigens present in breast milk ( molitor et al . , 2004 )
may generate nima - specific treg , which may prevent the deletion of maternal cells by nima - specific effector t cells , resulting in a high level of microchimerism .
( 2009 ) reported that exposure to nima both in utero and by breastfeeding appears to generate higher levels of maternal microchimerism than in utero exposure alone , and that the degree of microchimerism , correlates with a prolonged survival in maternal heart grafts .
predicting acute gvhd in vitro before transplantation has been tried in an hla - mismatched setting , but satisfactory methods had not been established .
the frequencies of cytotoxic t lymphocyte precursor ( ctlp ) and helper t lymphocyte precursor ( htlp ) cells , as well as mlr , were reported for the methods that had been evaluated to detect an individual s reactivity to nima in vitro ( falkenburg et al . , 1996 ; moretta et al . , 1999 ;
( 1999 ) described that the frequency of nima - specific ctlp in cord blood samples could be measured in order to better define the phenomenon of nima tolerance .
nima - reactive cord blood cells were detectable , but the authors of that study could not show a difference in the ctlp frequency toward nima and nipa .
neither the ctlp nor htlp frequencies against nipa were not significantly different from those against nima . indeed , kircher et al .
( 2004 ) showed that the ctlp and htlp frequencies were not predictive for the risk of acute gvhd in patients who received allogeneic hsct .
collectively , established test systems are not available for predicting an alloreaction and the outcome after hsct .
ctlp reflects the alloreactivity of class i mismatch , and mlr and htlp reflect alloreactivity of class ii mismatch .
they generated carboxyfluorescein diacetate succinimidyl ester - labeled cd4cd25 foxp3 cells in mlr , which they called  treg mlr ,  with varying hla disparities and cell components .
thus , all of the above - mentioned methods can detect mhc class i or class ii separately , but it is difficult to detect them simultaneously ( table 2 ) .
we recently , reported a novel method , mlr - elispot assay , that overcomes these disadvantages , as shown in figure 3a .
( a ) the elispot assay combined with mlr ( mlr - elispot ) is a sensitive functional assay to detect alloreactivity for both major and minor histocompatibility antigens in mice .
( b ) the mice were classified into two groups based on their reactivity to nima ; the high responders ( hr  mean  1 sd in nima - non - exposed ) or the low responders ( lr < mean  1 sd ) group by using mlr ( araki et al . , 2010 ) .
the ifn--producing ability before the induction of gvhd was presented by the mlr - elispot assay .
peripheral blood mononuclear cells from nima - exposed lr mice ( n = 8) , nima - exposed hr mice ( n = 7 ) , and non - exposed mice ( n = 6 ) were stimulated with b10 mouse peripheral blood mononuclear cells .
the data are expressed as the means  sd of individual animals . * p < 0.05 .
the alloreactivities of nima - exposed mice and nima - non - exposed mice were evaluated by mlr , and we found a wide range of reactivity ( araki et al . , 2010 ) .
this indicates that the fetomaternal interaction acts on both tolerance ( low - responder , lr ) and sensitization ( high - responder , hr ; molitor - dart et al . , 2008 ; van halteren et al . , 2009 ) .
( 2000 ) detected a reactivity to nima by mlr , and ctlp and htlp , respectively .
interestingly , when we scrutinized the figures in their articles , the individual reactivities of the nima - exposed group showed a wider range than the control group , and those reactivities seem to be divided into low and high reactions , although the authors of those studies did not discuss these observations .
this was a further indication that reactivity to nima could be detected in vitro , and that the fetomaternal interaction promoted either tolerance or sensitization .
recently , we demonstrated that the number of cells producing ifn- was significantly lower in the nima - exposed lr group than the hr group by using an mlr - elispot assay in a murine model ( figure 3b ) .
thus , the capacity for an individual to produce ifn- against allogeneic antigens or nima could differentiate lr from hr .
this assay is easily applicable in humans , and is a versatile method to detect reactivities to mhc class i , as well as class ii .
in other words , this assay might be useful to predict the total immunological reaction of donor t cells to the recipient in hla - mismatched hsct .
non - inherited maternal antigens - mismatched haploidentical hsct has been progressing , and now can lead to sustained engraftment , lower early treatment - related mortality , and acceptable rates of acute gvhd . however , it is difficult to predict severe acute gvhd prior to transplantation .
our recent report addressed this issue ( araki et al . , 2010 ) . the nima effect directed toward mhc antigens
there was an unevenness in the acquisition and maintenance of microchimerism in offspring , which was not due solely to differences in mhc gene inheritance .
although t cell replete haploidentical transplantation is performed only when there is positive microchimerism , the individual reactivity of the donor is not evaluated at present . therefore , our study is clinically relevant , and t cell replete nima - mismatched haploidentical transplantation can be performed more safely in the future by evaluating the responses of the ifn--producing cells of the donor against nima .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
 major histocompatibility complex antigens that provoke severe transplant reactions are referred to as the human leukocyte antigen ( hla ) in human and as the h-2 in mice . 
 even if the donor and recipient are hla - identical siblings , graft - versus - host reactions have been linked to differences in the minor histocompatibility antigen . as the chance of finding an hla - identical sibling donor is only 25% , 
 attention has been focused on using alternative donors . 
 an hla - mismatched donor with non - inherited maternal antigens ( nima ) is less immunogenic than that with non - inherited paternal antigens , because the contact between the immune systems of the mother and child during pregnancy affects the immune response of the child against nima . 
 however , the immunologic effects of developmental exposure to nima are heterogeneous , and can be either tolerogenic or immunogenic . 
 we recently have devised a novel method for predicting the tolerogenic effect of nima . in this review , 
 we overview the evidence for the existence of the nima tolerogenic effect , the possible cellular and molecular basis of the phenomenon , and its utilization in hematopoietic stem cell transplantation . 
 we suggest a future direction for the safe clinical use of this phenomenon , fetomaternal tolerance , in the transplantation field . 
INTRODUCTION
HISTOCOMPATIBILITY ANTIGENS IN HUMANS AND MICE
CLINICAL SIGNIFICANCE OF NON-INHERITED MATERNAL ANTIGEN
MURINE MODELS FOR MAJOR AND MINOR HISTOCOMPATIBILITY ANTIGENS TO NIMA
MATERNAL MICROCHIMERISM AND FETOMATERNAL TOLERANCE
PREDICTION OF ACUTE GVHD IN HLA-MISMATCHED HSCT
CONCLUSION
Conflict of Interest Statement