donor specific tolerance through mixed chimerism can be achieved in various animal models by nonmyeloablative bmt and cb .
success rates of chimerism and tolerance induction have typically been high when donor - recipient strain combinations were used which cross only mhc barriers but share the same mhag background [ 2 , 3 ] .
however , this setting does not reflect the clinical situation where mhag disparities exist universally . when switching to a different donor - recipient combination , which crosses mhc plus multiple mhag barriers ( balb / c  bl6 ) , retrospective review of pooled results revealed that overall only approximately 75% of mice developed lasting chimerism and of those that became chimeric approximately 15% rejected donor type skin grafts [ 47 ] .
mhag are polymorphic non - mhc proteins that are able to induce a t cell response due to allelic variations between donor and recipient .
mhag play a prominent role in hematopoietic stem cell transplantation ( hsct ) in many regards .
striking differences in engraftment of purified hematopoietic stem cells and/or development and severity of gvhd depending on mhag disparities were found in murine mhc - matched donor host strain combinations . in the human setting ,
disparities in mhag increase not only the rates of rejection and graft - versus - host - disease ( gvhd )   but also the efficacy of graft - versus - leukemia - effects ( gvl ) .
recently , mhag have also been reported to play an essential role in the persistence of donor chimerism .
skin allografts differing only in minor antigens are rejected with the same pace as mhc disparate allografts . moreover , a single mhag disparity was sufficient to induce chronic rejection of cardiac allografts in a congenic mouse model . in clinical studies of kidney transplantation
only limited and controversial data exist regarding the impact of mhag disparities on graft survival .
humoral immunity to specific mhag , such as antibodies to angiotensin type 1 receptor ( at1r ) and endothelin type a receptor ( etar ) , have been shown to correlate with an increased incidence of acute rejection and inferior long - term graft survival in kidney and heart allografts .
however , so far the role of mhag disparities in the induction and maintenance of tolerance through mixed chimerism has not been clearly defined .
therefore , we investigated different donor host strain combinations displaying mhc mismatches only ( either h2 or h2 ) or mhc plus multiple mhag mismatches ( h2 ) .
female c57bl/6 ( bl6 : h2 ) , balb / c ( h2 ) , and c3h / n ( h2 ) mice were obtained from charles river laboratories ( sulzfeld , germany ) and b10.d2 ( h2 ) and b10.a ( h2 ) from the jackson laboratory ( bar harbor , me ) .
all mice were housed under specific pathogen free conditions and used between 8 and 10 weeks of age .
all experiments were approved by the local review board of the medical university of vienna and were performed in accordance with national and international guidelines of laboratory animal care .
age - matched female bl6 recipients underwent nonmyeloablative total body irradiation ( tbi , 3  gy , d  1 ) prior to the intravenous injection of approximately 2  10 unseparated bone marrow cells ( bmc ) from balb / c , b10.d2 , or b10.a donors as previously described [ 46 ] .
additionally mice were injected intraperitoneally with an anti - cd154 mab ( mr1 ; 1  mg d0 ) and hctla4ig ( 0.5  mg d+2 ) .
( new hampshire , usa ) and hctla4ig was generously provided by bristol - myers squibb pharmaceuticals ( princeton , new jersey ) .
full thickness tail skin from sacrificed balb / c , b10.d2 , or b10.a mice , respectively ( donor specific ) , and c3h / n ( h2 ; 3rd party ) was grafted 7 or 15 weeks after bmt .
recipient mice were anesthetized through intraperitoneal injection of a mixture of ketamine ( 100  mg / kg ) and xylazine ( 5  mg / kg ) before attachment of skin grafts at the lateral thoracic wall .
two - color fcm was used to distinguish donor and recipient cells of particular lineages , by staining with fluorescein isothiocyanate- ( fitc- ) conjugated antibodies against cd4 , cd8 , b220 , and mac1 and a biotinylated antibody against h-2d ( 34 - 2 - 12 , developed with phycoerythrin streptavidin ) and irrelevant isotype controls . to analyze the expression of v-subunits staining
was performed with fitc - conjugated antibodies against v8.1/2 and v11 and pe - conjugated antibodies against cd4 .
mice were considered chimeric if they showed detectable donor cells within the myeloid lineage plus at least one lymphoid lineage .
an epics xl - mcl flow cytometer ( beckman coulter , il alliance , vienna , austria ) was used for acquisition and expo32 adc software , applied cytometry systems , was used for analysis of flow cytometric data .
four micrometer sections were cut from paraffin - embedded tissue fixed in 4.5% formalin ( ph of 7.5 ) , stained with hematoxylin - eosin and giemsa according to standard protocols , and analyzed by an experienced pathologist in blinded fashion according to the banff 2007 working classification of skin - containing composite tissue allograft pathology .
a two - tailed student 's t - test was used for comparing percentages of v-positive populations and levels of chimerism within several cell lineages .
skin graft survival was calculated according to the kaplan - meier product limit method and compared between groups by using the log - rank test .
the fisher exact test was used to compare histologically categorized skin grafts   of different donor groups . a p value less than 0.05 was considered to be statistically significant .
three groups of mice were treated with a previously published nonmyeloablative bmt protocol [ 46 ] .
bl6 recipients ( h2 ) received 2  10 bmc from different donor strains with mismatches of mhc with / without additional mhag mismatches after 3  gy tbi ( d  1 ) together with cb consisting of a single dose each of anti - cd154mab ( 1  mg mr1 , d0 ) and ctla4ig ( 0.5  mg , d2 ) .
balb / c ( h2 ) , b10.d2 ( h2 , same background as bl6 ) , and b10.a ( h2 , same background as bl6 ) mice were used as donors ( figure 1(a ) ) to investigate the potential influence of mhag on top of the burden of mhc mismatch and the influence of the specific mhc haplotype on the induction of long - term multilineage chimerism ( h2 versus h2 ) . in bl6 recipients of balb
/ c bmc high levels of multilineage chimerism ( tested in cd4 , cd8 , b cells , and myeloid cells , figure 1(b ) ) were initially induced in 17 of 18 mice . at 16 weeks after bmt only 13 of 18 mice stayed chimeric ( pooled data of two independent experiments ) .
this result is consistent with numerous previous experiments using this protocol showing that chimerism is lost in approximately 25% of recipients over time .
in contrast , 21 of 21 bl6 mice receiving bmc of b10.d2 developed multilineage chimerism which remained stable in all but one animal over time ( p < 0.05 versus balb / c after week 16 after bmt , figure 1(c ) ) . interestingly the rate of chimeras also dropped in bl6 recipientsof b10.a bmc from initially 17 of 20 immediately after bmt to 13 of 20 at week 16 ( p < 0.05 versus b10.d2 , figure 1(c ) ) .
these data indicate that mhag mismatches pose a barrier to establishing long lasting multilineage chimerism through bmt with cb .
additionally the mhc haplotype ( h2 versus h2 ) also seems to influence bone marrow ( bm ) engraftment with this cb - based nonmyeloablative protocol . analyzing lineage - specific blood chimerism levels in successful long - term chimeras it was noted that b cell chimerism was significantly higher in recipients of b10.d2 bmc compared to recipients of balb / c bmc at each investigated time point ( e.g. , b10.d2 versus balb / c : 81.9%  7.2 versus 61.3%  9.4 at week 19 , figure 1(d ) , p < 0.01 , p < 0.05 ) , whereas cd8 and myeloid cell chimerism levels were comparable between these two groups .
cd4 chimerism was significantly higher in recipients of balb / c bmc at week 19 after bmt ( b10.d2 versus balb / c : 36.08%  14.53 versus 58.75%  12.13 , figure 1(d ) , p < 0.01 ) .
significantly lower chimerism levels were observed in myeloid lineages in b10.a bmc recipients compared to recipients of balb / c bmc from week 6 on .
no consistent differences in t cell chimerism ( cd4- and cd8 cells ) and b cell chimerism levels were observed between long - term chimeras after bmt from b10.a or balb / c donors .
nonetheless , cd4 and cd8 t cell chimerism levels were significantly higher ( b10.a versus balb / c : cd4 : 25.56  10.43 versus 13.51  4.82 and cd8 : 24.55  7.83 versus 15.48  8.86 ) in recipients of b10.a bmc 6 weeks after bmt but declined below that of balb / c bmc recipients by week 19 ( b10.a versus balb / c : cd4 : 37.71  25.44 versus 58.75  12.13 , p < 0.05 ) .
the total level of donor chimerism among leukocytes was 62.41% versus 62.26% versus 53.41% in recipients of balb / c versus b10.d2 versus b10.a bone marrow , respectively ( at 19  weeks after bmt ) .
these results suggest that both individual mhc haplotype and minor antigen disparities influence the degree of chimerism in distinct lineages .
specific skin graft acceptance is considered as a stringent test to indicate transplantation tolerance . to investigate a possible influence of mhag mismatches on skin graft acceptance
, donor and 3rd party tail skin was transplanted 2 - 3 months after bmt . among successfully established chimeras long - term donor skin graft survival ( > 130 days )
this rate is similar to our previous experience with this protocol [ 4 , 6 , 7 ] .
in contrast , recipients of b10.d2 or b10.a bmc showed a significantly better long - term donor skin graft survival ( b10.d2 : 16/17 , b10.a : 12/12 , figure 2(a ) , p < 0.05 for balb / c versus b10.d2 and b10.a donors ; pooled data of two independent experiments ) .
however , in recipients of balb / c bmc no significant difference in chimerism levels of long - term chimeras , which rejected donor skin grafts in comparison to tolerant animals was observed ( figure 2(b ) ) . regarding the macroscopic appearance of donor skin grafts of long - term chimeras , no shrinking , thickening , or loss of surface aspect
was observed in mhag matched b10.d2 and b10.a donor grafts in contrast to balb / c grafts ( figure 2(b ) ) .
histological analysis of those donor grafts that were retained until the end of follow - up revealed that balb / c donor grafts exhibited signs of chronic rejection , like sparse infiltration with lymphocytes and mast cells together with focally dense lymphocytic - mononuclear cell infiltration . in comparison no incidence of dense focal lymphocytic infiltration was seen , in b10.d2 donor grafts ( figure 2(c ) ) .
skin grafts were scored by a blinded pathologist according to the banff 2007 working classification of skin - containing composite tissue allograft pathology .
moderate ( grade 2 ; 2/3 ) and mild signs of inflammation ( grade 1 ; 1/3 ) were found in balb / c grafts , whereas no signs of inflammation ( grade 0 ) were seen in 4/4 b10.d2 grafts ( p < 0.05 versus balb / c donors ) and 2/4 b10.a grafts ( 2/4 grade 1 , p = n.s . versus balb / c , figure 2(d ) ) . taken together , these data suggest that mhag disparities increase the rate of chronic skin graft rejection in recipients with persistent levels of mixed chimerism .
peripheral and central clonal deletion are important mechanisms for the induction and maintenance of tolerance in models of mixed chimerism induced by bmt plus cb .
notably not all mice become chimeric with such a protocol and not all chimeric mice accept donor skin grafts indefinitely .
we followed v11 + cd4 + t cells , which in this model recognize endogenous superantigens presented by donor mhc ii ( i - e ) but not recipient mhc class ii and thus serve as surrogate markers for donor - alloreactive t cells . like in previous experiments
balb / c long - term chimeras displayed marked deletion of v11 + cd4 + t cells by week 4 after bmt ( 2.04%  0.80 [ n = 8 ] versus 5.20%  0.17 in nave bl6 mice [ n = 2 ] )
. compared to balb / c chimeras , a similar degree of deletion was observed in b10.d2 chimeras ( 1.99%  0.69 [ n = 11 ] , p = n.s . ) .
interestingly , transplantation of b10.a ( h2 , mhag matched ) bone marrow led to a significantly more pronounced early deletion compared to balb / c and b10.d2 ( 0.67%  1.62 [ n = 9 ] , p < 0.05 versus balb / c and b10.d2 donors , both lineages ) . in contrast irrelevant v8.1/2 + cd4 + t cells were not deleted in either group after bmt , indicating the specificity of the deletion for superantigens presented by the donor ( figure 3(a ) ) .
ten weeks after bmt the degree of deletion was significantly enhanced in long time chimeras after balb / c and b10.d2 bmt ( 0.93%  0.22 [ n = 8 ] and 1.26%  0.27
[ n = 11 ] , p < 0.01 versus week 4 for both lineages ) but still was significantly less pronounced than in recipients of b10.a bmc ( 0.23%  0.16 [ n = 9 ] , p < 0.01 versus balb / c and b10.d2 , both lineages ; figure 3(b ) ) .
concluding , the type of mhc ( i.e. i - e versus i - e ) may influence the extent and kinetic of deletion of donor - reactive t cells .
in this study we provide evidence that mhag disparities play a decisive role in the induction and maintenance of tolerance in recipients conditioned with nonmyeloablative bmt and cb , in which mhag both impede the engraftment of bm and promote the rejection of donor skin in successfully established mixed chimeras .
tolerance induction through donor bmt depends on two , mechanistically separate , events : successful engraftment of donor bm and durable tolerization of all relevant donor antigens in the context of donor chimerism .
the frequency of successful long - term chimeras was higher in the absence of mhag disparities when the mhc haplotype was the same ( 13/18 for balb / c versus 20/21 for b10.d2 ) .
these results are in line with findings that mhc - matched bm is rejected in sublethally irradiated mice .
only some of the minor antigens that drive alloreactivity in this strain combination ( balb / c to bl6 ) have been identified , with h60 provoking a particularly strong reaction .
h60 is a ligand for the activating receptor nkg2d which is expressed on nk cells and on activated cd8 t cells , which are both effective mediators of allogeneic bm rejection .
intriguingly , balb / c donor mice feature a higher surface expression of h60 than recipient bl6 mice .
nkg2d has been reported to enhance nk cell mediated bm rejection in semiallogeneic pairs ( balb / c to f1 )   but seems to be dispensable in fully mismatched combinations ( balb / c to bl6 ) . alternatively , nkg2d can function as a costimulatory receptor to augment the response of nave cd8 t cells .
this could provide an alternative costimulatory route for cd8 t cells when cd40 and cd28 pathways are blocked .
the role of nkg2d in cd8 t cell mediated bm rejection has not yet been evaluated although it is well established that h60 triggers an abnormally high number of responding cd8 t cells .
unexpectedly , significantly fewer mice developed long lasting multilineage chimerism after receiving bm from b10.a compared to b10.d2 donors ( both mhag matched to recipient ) . since the only difference between b10.a and b10.d2 is the h2 haplotype these results suggest that the mhc haplotype per se influences chimerism induction .
distinct h2 haplotypes are known to stimulate varying numbers of alloreactive t cells and thus exhibit a varying degree of immunogenicity , which apparently also influences bm rejection versus engraftment .
interestingly , if engraftment is successful , b10.a chimeras accepted donor skin grafts to a comparable extent as b10.d2 chimeras without macroscopical and histological signs of chronic inflammation .
thus , distinct mhc haplotypes impede bm engraftment to varying degrees but do not affect the success of skin graft tolerance in established mixed chimeras . with regard to the second event
, mhag disparities increased the rate of skin graft rejection in successfully established chimeras ( 10/13 for balb / c versus 28/29 for b10.d2 and b10.a ) .
in addition , the surviving skin grafts of balb / c but not b10.d2 donors exhibited histological signs of chronic inflammation . thus , mhag disparities can drive chronic rejection in the presence of stable mixed chimerism .
several groups attributed tissue specific antigens , which are not present in the bm but the skin , for this state of so - called  split tolerance  . obviously , for clinical translation this hurdle of mhag disparities with its associated risk of  split tolerance 
recently , we could demonstrate that the combination of regulatory cell therapy with donor bm transplantation leads to a state of tolerance that encompasses donor mhag .
critically , this regimen relies on extensive regulatory mechanisms , including linked suppression , that appear superior and indeed indispensable for tolerization of donor mhag ( pilat et al .
this study reveals that mhag disparities have a negative impact on bm engraftment and tolerance maintenance in a nonmyeloablative , cb - based chimerism model .
preclinical tolerance protocols should encompass mhag disparities to reflect the clinical setting and need to induce mechanisms capable of durable tolerization of donor mhag .
 mixed chimerism and tolerance can be successfully induced in rodents through allogeneic bone marrow transplantation ( bmt ) with costimulation blockade ( cb ) , but varying success rates have been reported with distinct models and protocols . 
 we therefore investigated the impact of minor antigen disparities on the induction of mixed chimerism and tolerance . 
 c57bl/6 ( h2b ) mice received nonmyeloablative total body irradiation ( 3  gy ) , costimulation blockade ( anti - cd40l mab and ctla4ig ) , and 2  107 bone marrow cells ( bmc ) from either of three donor strains : balb / c ( h2d ) ( mhc plus multiple minor histocompatibility antigen ( mhag ) mismatched ) , b10.d2 ( h2d ) or b10.a ( h2a ) ( both mhc mismatched , but mhag matched ) . 
 macrochimerism was followed over time by flow cytometry and tolerance was tested by skin grafting . 
 20 of 21 recipients of b10.d2 bmc but only 13 of 18 of balb / c bmc and 13 of 20 of b10.a bmc developed stable long - term multilineage chimerism ( p < 0.05 for each donor strain versus b10.d2 ) . 
 significantly superior donor skin graft survival was observed in successfully established long - term chimeras after mhag matched bmt compared to mhag mismatched bmt ( p < 0.05 ) . 
 both minor and major antigen disparities pose a substantial barrier for the induction of chimerism while the maintenance of tolerance after nonmyeloablative bmt and costimulation blockade is negatively influenced by minor antigen disparities .     