
 human leukocyte antigen ( hla ) matching significantly reduces the risk of graft rejection and graft failure after solid - organ transplantation [ 13 ] and graft - versus - host disease ( gvhd ) after hematopoietic stem - cell transplantation ( hsct ) [ 49 ] .
these pathological conditions evolve due to an alloreactive immune response that is initiated through interaction of allogeneic hla with antibodies or the t - cell receptor ( tcr ) .
the subsequent immune response directed against allogeneic hla impairs transplant outcome , emphasizing the need to avoid alloreactive responses after transplantation .
the highly polymorphic hla system can be subdivided into two major classical classes : hla class i and hla class ii .
in general , hla class - i molecules ( hla - a , -b , and -c ) present endogenous peptides of 811 amino acids in length that can be recognized by cd8 + t cells , while hla class - ii molecules ( hla - dr , -dq , and -dp ) present exogenous peptides of 1318 amino acids in length that can be recognized by cd4 + t cells .
hla class - i molecules consist of a polymorphic alpha chain and a nonpolymorphic beta-2-microglobulin and have a rather closed peptide binding groove .
on the other hand , hla class - ii molecules consist of a polymorphic alpha and beta chain and have a more open structure .
acquiring hla - matched donors for transplantation is very challenging , due to the high level of polymorphisms in the hla system .
hla incompatible transplantations can therefore not be avoided for a large number of patients . in those cases where a fully hla - matched donor is not available
, there is a clinical need to predict whether a certain hla mismatch will elicit severe b - cell and t - cell - mediated alloreactive responses or not .
there is cumulating evidence that these high - risk hla mismatches ( so - called nonpermissible mismatches / unacceptable mismatches ) and well - tolerated hla mismatches ( so - called permissible mismatches / acceptable mismatches ) exist , as epidemiological studies have shown that permissibility of hla - mismatched combinations is highly variable [ 6 , 7 , 10 ] .
for example , hla - b44:02 and hla - b44:03 mismatching leads to the induction of allospecific cd8 + t cells in vitro   and bone marrow - allograft rejection in vivo .
the amino - acid sequences of hla - b44:02 and hla - b44:03 differ only in one amino acid , indicating that even small amino - acid changes between hla molecules can result in major alloreactive immune responses after transplantation . on the other hand ,
hla class - i mismatches that are highly diverse may well be tolerated in hsct .
differences in permissibility between hla - mismatched combinations may be explained by a different impact of amino - acid polymorphisms on peptide - binding features .
some amino - acid sequence polymorphisms will alter peptide - binding motifs and peptide - hla complex conformation , thereby potentially inducing alloreactive immune responses , while others will not alter peptide - hla landscapes .
characterizing the permissibility of hla mismatches prior to transplantation allows selection of the most optimal donor - recipient match and thereby will help to diminish the risk of posttransplantation complications after hla incompatible transplantations .
however , epidemiological studies do not provide a universal tool for defining permissibility for every hla - mismatched combination , as these data are limited to the specific hla - mismatched combinations studied ; very large study populations would be required to study all potential combinations .
several approaches have therefore been developed to define permissibility of hla - mismatched combinations ; some of these approaches are very useful in predicting alloreactivity .
we here review the current knowledge regarding hla - directed alloreactivity and the various in vitro and in silico methodsthat can be used to predict this alloreactivity .
hla alloreactivity in transplantation involves both b - cell- and t - cell - mediated responses .
three mechanisms of alloreactivity directed towards allogeneic hla have been described : direct , indirect , and semidirect allorecognition .
igg hla alloantibodies directly recognize intact allogeneic hla molecules that are present on the cell surface .
these antibodies play a pivotal role in solid - organ transplantation and probably also have a role in hsct [ 1518 ] . the humoral response directed against allogeneic hla
can be established upon exposure to allogeneic hla during pregnancies , blood transfusions , or ( previous ) transplantations . during this response
these peptides can subsequently be presented on hla class - ii molecules that are present on the cell surface .
recognition of these hla class - ii presented hla - derived epitopes by cd4 + t cells results in b - cell activation and igm to igg isotype switching .
donor - specific igg hla antibodies ( dsa ) that are subsequently produced can bind directly to small polymorphic amino - acid residue patches that are present on the molecular surface of hla antigens [ 2022 ] , thereby inducing rejection of graft tissue / cells , designated as antibody - mediated rejection .
in addition to alloantibodies , alloreactive t cells can also directly recognize intact allogeneic hla molecules .
there is compelling evidence that cross - reactive t cells are involved in direct t - cell recognition [ 2431 ] .
these cross - reactive t cells initially react towards a foreign peptide , for instance , a viral peptide , presented by self - hla .
however , these t cells can also respond to allogeneic hla presenting a self- or viral peptide [ 2431 ] .
although these cross - reactive t cells can persist over time , direct t - cell recognition is predominantly involved in the acute stage of alloreactivity .
intact hla molecules present on resident donor - derived antigen - presenting dendritic cells are considered to be the driving force behind direct recognition in solid - organ transplantation , since parenchymal cells within transplanted tissues are unable to induce direct t - cell recognition ( reviewed in ) . because these dendritic cells are depleted over time , the contribution of direct recognition in chronic graft rejection after solid - organ transplantation is limited [ 32 , 33 ] . in contrast to direct t - cell recognition , indirect t - cell recognition is considered to be mainly involved in later stages of alloreactivity . during indirect recognition ,
t cells recognize processed epitopes derived from allogeneic hla that are presented by hla molecules that are likely shared between donor and recipient [ 35 , 36 ] , as t cells are restricted to self - hla .
indirect t - cell recognition is also involved in the formation of hla alloantibodies , since t - cell recognition of b - cell presented hla epitopes is required in this process [ 19 , 37 ] .
thus , indirect t - cell recognition may also partly contribute to early alloreactivity , as indirect recognition can amplify the direct recognition response .
in semidirect allorecognition , allogeneic hla : peptide complexes are transferred from allogeneic cells to autologous dendritic cells , resulting in a chimeric antigen - presenting cell .
transfer of allogeneic hla : peptide complexes can be achieved through secretion of endosomes containing hla : peptide complexes   or through cell - to - cell contact between donor and recipient dendritic cells .
antigen - presenting cells that acquire intact allogeneic hla : peptide complexes on their cell surface may elicit both direct and indirect alloreactive t - cell responses .
although in vivo evidence for the role of the semidirect allorecognition pathway in graft rejection and gvhd is limited , it has been shown that this pathway is able to elicit cytotoxic alloimmunity in vitro and in vivo   and that the transfer of allogeneic hla : peptide complexes likely occurs in an in vitro system of gvhd .
humoral sensitization to hla class - i and class - ii epitopes and the subsequent production of hla - specific antibodies can occur upon pretransplant exposure to allogeneic hla .
the presence of dsa before transplantation is related to antibody - mediated rejection and significantly impairs graft prognosis [ 15 , 16 ]
. therefore , evaluation of hla - sensitizing events ( i.e. , pregnancies , blood transfusions , and previous transplants ) is generally included in standard pretransplantation screening .
pregnancy is a major contributor to hla sensitization , as approximately 30% of the pregnancies results in child - specific sensitization towards hla - a , -b , -c , and/or -dr loci .
moreover , the hla sensitization frequency increases with the number of full - term pregnancies .
blood transfusions can induce hla sensitization in approximately a third of the solid - organ transplantation recipients . however , blood transfusions have a less prominent effect on hla alloimmunization than pregnancy and solid - organ transplantation [ 44 , 45 ] .
in addition to the classical sensitizing events , hla alloantibodies can also be raised against epitopes in allergens , ingested proteins , and microorganisms that are cross - reacting with hla .
although the presence of these  natural  dsa in kidney recipients is associated with the induction of mild episodes of antibody - mediated rejection , these patients have favorable graft outcome .
therefore , the existence of these  natural  dsa prior to transplantation is currently not a contraindication for transplantation .
although dsa detection methods are important tools for risk assessment prior to transplantation , pretransplant evaluation of preformed dsa remains challenging .
for example , antibodies might become undetectable at the moment of transplantation due to the decay of antibody levels over time .
the clinical relevance of these preexisting low dsa levels is highly variable ; some preformed dsa will elicit hla alloreactivity in vivo , whereas others will not .
currently used detection methods may thus not detect the whole repertoire of clinically relevant dsa .
the complement - dependent cytotoxicity ( cdc ) crossmatch assay ( reviewed in ) is a potent manner to measure the presence of clinically relevant antibodies , whereas other dsa detection assays , like the hla - based enzyme - linked immunoabsorbent assay ( elisa ) and luminex - based assays [ 50 , 51 ] , provide valuable but limited information about the clinical relevance of identified dsa .
currently , the cdc assay seems to be a potent indicator for alloreactivity , while in vitro dsa detection methods can further support the matching procedure for solid - organ transplantation .
combining in vitro assays with an in silico prediction method allows identification of acceptable hla mismatches towards which a recipient will likely not develop antibody - mediated responses .
assessment of humoral sensitization to allogeneic hla was initially performed by the cdc crossmatch assay .
this assay measures the presence of preformed or de novo formed antibodies through their induction of complement - dependent lymphocyte killing .
a positive cdc test was associated with a significantly impaired outcome after kidney transplantation [ 49 , 52 ] . despite its potency to mimic the in vivo situation , cdc crossmatch assays lack sensitivity   and may show false positive results . to overcome these problems ,
a more sensitive assay was developed : the flow cytometry - based crossmatch ( fcxm ) assay .
however , both fcxm and the classical cdc crossmatch test correlate equally well to clinical outcome after kidney transplantation .
the lack of sensitivity and specificity of cytotoxicity crossmatch assays has led to development of solid - phase assays , such as the hla - based elisa and luminex assays [ 50 , 51 ] .
these solid - phase methods , particularly luminex , are very sensitive and specific ; relevant anti - hla class - i and class - ii antibody profiles in solid - organ transplant recipients can be identified and monitored over time . combining antibody profiles that are present in solid - organ transplant recipients , with hla typing of the donor , designated as virtual crossmatching , allows identification of dsa and therefore might be useful in risk stratification prior to solid - organ transplantation ( reviewed in [ 56 , 57 ] ) .
unfortunately , estimation of the clinical relevance of dsa detected with solid - phase assays remains challenging , as tools to discriminate between nondetrimental dsa and deleterious dsa are lacking .
nevertheless , the presence of class - i and class - ii dsa detected by luminex in the absence of positive cdc assay is suggested to be indicative for impaired graft outcome in kidney transplantation . 
 in vitro cdc - based dsa detection assays
have their limitations ; these assays are not suitable to determine hla - mismatch permissibility for highly sensitized transplantation candidates . because of the high sensitization levels in those individuals , cdc assays often become almost completely positive , which complicates selection of suitable cdc - negative donors that will not elicit hla alloreactivity in vivo .
therefore , alternative in silico methods were sought to predict acceptability of hla - mismatched combinations .
the in silico algorithm hlamatchmaker is based on the principle that hla - specific alloantibodies can bind to distinct amino - acid polymorphisms ( immunogenic epitopes ) present on hla antigens [ 20 , 21 ] .
multiple polymorphic amino - acid residues on the molecular surface of hla antigens have been identified .
some of these residues are inaccessible for antibodies , since they are located near the cell membrane or within peptide - binding groove of the hla molecule , while other residues are fully accessible for antibodies [ 20 , 21 ] .
hlamatchmaker uses this knowledge to predict which hla mismatches are not able to induce complications in transplantation recipients by defining the acceptable mismatches [ 20 , 21 ] . initially ,
hlamatchmaker defined immunogenic epitopes as antibody - accessible , linear sequences of amino - acid polymorphisms ( triplets ) [ 20 , 21 ] .
triplets that are present in donor hla antigens , but not in the recipient hla antigens , were considered to elicit humoral responses [ 20 , 21 ] . on the other hand , triplets that are present in both donor and recipient hla antigens were considered as acceptable [ 20 , 21 ] .
thus , hlamatchmaker provides a tool for identification of acceptable hla mismatches . the clinical applicability of hlamatchmaker in matching strategies has been extensively evaluated .
it has been shown that the triplet version of hlamatchmaker is a potent indicator for the presence and magnitude of allogeneic hla - directed antibody responses in renal transplantation and during pregnancy [ 59 , 60 ] .
in contrast , the number of triplet mismatches was not indicative for the induction of t - cell alloreactivity .
this lack of correlation between triplets and t - cell alloreactivity is probably caused by alternative epitope binding by t cells or by the involvement of larger polymorphic sequences in t - cell alloreactivity . with regard to hsct
, the number of triplets did not correlate to acute gvhd , engraftment , or survival . despite its applicability in hla - matching strategies ,
the triplet version of hlamatchmaker represents an incomplete repertoire of immunogenic epitopes , as only linear sequence positions are implemented .
this hiatus has resulted in the development of a redefined version of hlamatchmaker that identifies eplets .
eplets are immunogenic hla epitopes that are critical for antibody binding and consist of polymorphic amino - acid patches located at the molecular surface of hla molecules .
these polymorphic patches may consist of polymorphisms in linear sequence positions and three - dimensional polymorphic patches in discontinuous sequence positions .
therefore , implementation of eplets into the algorithm has led to a more accurate definition of structural hla epitopes .
evaluation of the eplet version of hlamatchmaker has shown a similar performance in predicting allogeneic hla acceptability compared to the triplet version .
nevertheless , the eplet version of hlamatchmaker provides further discrimination of highly divergent hla specificities .
although conflicting results were reported with regard to the prognostic information that is provided by hlamatchmaker on graft outcome ( [ 6366 ] , reviewed in ) , it is generally accepted that hlamatchmaker is a suitable tool to analyze serum antibodies and to identify acceptable mismatches in solid - organ transplantation . however , hlamatchmaker is inappropriate for hsct donor selection .
in addition to the number of eplets as determined by hlamatchmaker , additional determinants can be used to define allogeneic hla acceptability , for instance , physiochemical properties of polymorphic amino acids .
differences in physiochemical properties between mismatches , including electrostatic potential and hydrophobicity , are useful to predict hla class - i- and class - ii - specific alloantibody responses prior to solid - organ transplantation [ 6870 ] . with higher physiochemical disparity between hla mismatches , the risk of antibody development increases after kidney transplantation [ 6870 ] .
these observations suggest that differences in physiochemical properties between polymorphic amino acids may be relevant in defining acceptable hla mismatches .
the presence of t - cells directly recognizing intact allogeneic hla molecules was previously shown in individuals suffering from graft rejection after solid - organ transplantation [ 71 , 72 ] and gvhd after hsct .
there is compelling evidence that direct t - cell alloreactivity results from cross - reactive t cells that are initially primed by a foreign peptide , for instance , a viral peptide [ 2431 ] .
for example , the hla - b08:01-presented ebv peptide flrgraygl is recognized by an ebv - specific tcr , that possesses cross - reactive capacities towards hla - b44:02-presented peptide eeyqafty [ 24 , 75 ] .
thus , the hla - b08:01-presented peptide flrgraygl elicits a public immune response . during a public immune response , the immune response directed against an identical epitope
virus - specific t cells can be detected in high levels in healthy individuals , it is likely that cross - reactive virus - specific t cells may be present in both solid - organ transplantation recipients and hsct donors prior to transplantation .
the presence of virus - specific t cells that are cross - reactive with allogeneic hla in these individuals may significantly contribute to complications after transplantation .
however , most virus - specific t - cell responses do not have the propensity to induce public tcr responses nor predictable cross - reactivity with allogeneic hla .
a single viral infection can therefore result in the establishment of multiple t cells that are cross - reactive to multiple hla molecules , whereas other viral infections do not give rise to these cross - reactive t cells .
in addition , virus - specific t cells with the same antigen specificity , but different tcrs , elicit different unpredictable patterns of alloreactivity [ 26 , 79 ] .
the molecular mechanism behind t - cell cross - reactivity is complex and currently incompletely understood .
t - cell cross - reactivity assumably arises due to structural homology of hla : peptide complexes ( reviewed in ) rather than sequence homology of the presented peptides . despite their sequence dissimilarity ,
the structure of flrgraygl and eeyqafty epitopes in the context of their presenting hla molecules is quite similar .
therefore , molecular mimicry likely attributes to the observed cross - reactivity between these epitopes . on the other hand ,
cross - reactive tcr in mice can dock to self - mhc : peptide complexes in a different orientation than to allogeneic mhc : peptide complexes , suggesting that cross - reactivity can be established without molecular mimicry .
thus , direct alloreactivity is a complex immune response that can only partially be explained by molecular mimicry . since the molecular mechanism behind direct t - cell recognition is poorly understood , prediction of alloreactivity based on viral history is complex .
knowledge about viral history is therefore not sufficient to predict direct t - cell alloreactivity directed towards allogeneic hla . since
direct t - cell allorecognition was studied intensively over the past decades , several alternative approaches to predict direct t - cell alloreactivity in vitro and in silico have been developed .
cytotoxic t lymphocyte precursor assays ( ctlp ) determine permissibility of hla mismatches through in vitro evaluation of effector cytotoxic t - cell induction .
this chromium 51 ( cr ) release - based assay , initially described by brunner et al .
further development of this assay has resulted in an assay that estimates the extent of alloreactive t - cell responses directed towards allogeneic hla . since
individual allogeneic hlas can be linked to ctlp frequencies , these assays are a useful approach to distinguish between permissible and nonpermissible mismatches in vitro .
more importantly , a high ctlp frequency correlates reasonably well with clinical outcome in vivo ; high ctlp frequencies were associated with graft rejection after solid - organ and tissue transplantation [ 73 , 84 , 85 ] and with gvhd and impaired survival after allogeneic hsct [ 82 , 83 , 86 ] .
association between graft failure and the presence of primed cytotoxic t lymphocytes in sensitized transplant candidates ( e.g. , women after previous pregnancy ) was shown as well . despite the usefulness of ctlp assay in estimating t - cell alloreactivity ,
the time - consuming and laborious character of this assay is a major drawback . in order to overcome these disadvantages , alternative in silico approaches
amino - acid polymorphisms at tcr - recognition and peptide - binding regions between hla class - i mismatches were analyzed for their physiochemical and/or position characteristics and were correlated to ctlp outcome .
these analyses resulted in the establishment of a novel algorithm , which aims to predict hla class - i mismatch - specific ctl alloreactivity .
although the algorithm can predict ctlp outcome reasonably well , usage of this model for donor selection seems limited ; this tool does not predict gvhd development in patients receiving hsct . the first clinically relevant model that successfully estimates the effect of direct recognition in hsct has recently been developed .
this hla - dpb1-restricted model is designated as the t - cell epitope ( tce ) model .
this model has been based on in vitro data from two alloreactive t - cell clones isolated from an hsct patient with graft rejection due to an hla - dpb1 mismatched graft .
membrane - bound intact hla was essential for recognition of the hla - dpb1 mismatch by the alloreactive t - cell clones ; the clones did not respond to b - lymphoblastoid cell lines transduced with a truncated mismatched - hla - dpb1 construct that did not lead to cell - surface expression of hla - dpb1 .
thus , it seems likely that these two alloreactive t - cell clones recognized the hla - dpb1 mismatched antigen in a direct manner . in order to identify patterns of recognition of other alleles
, the t - cell clones were further tested for their recognition of other hla - dpb1 alleles .
alleles were divided into three different immunogenic levels : highly immunogenic ( i.e. , both clones recognized the alleles ) , intermediate immunogenic ( i.e. , one of the clones recognized the allele but the other did not ) , or nonimmunogenic ( i.e. , both clones did not recognize the allele ) . since testing of all hla - dpb1 alleles in vitro is very time consuming , immunogenicity of other hla - dpb1 alleles was extrapolated , based on similarities between the peptide - binding grooves of the in vitro tested alleles and the not - tested hla - dpb1 alleles .
subsequently , hla - dpb1 mismatches were labeled as permissive or nonpermissive based on their immunogenic level and the concept of thymic education .
for example , when the hla - dpb1 allele of the donor belongs to the highly immunogenic group , then donor t cells should be educated not to respond to hla - dpb1 alleles belonging to the highly immunogenic group and , in theory , will also not respond to lower immunogenic alleles .
therefore , when the recipient has an hla - dpb1 allele belonging to the same or a lower immunogenic group , then the hla - dpb1 mismatch will be permissive in the graft - versus - host ( gvh ) direction . on the other hand ,
since the hla - dpb1 allele of the recipient is not immunogenic , recipient t cells are able to respond to ( higher ) immunogenic alleles of the donor .
thus , such mismatches are nonpermissive in the host - versus - graft ( hvg ) direction .
nonpermissive mismatches , defined by the tce model , are highly correlated to alloreactivity as reflected by gvhd , graft rejection , and transplant - related mortality after hla - dpb1-mismatched hsct [ 4 , 9092 ] .
counterintuitively , in these situations , the direction of the nonpermissiveness appears not to be important : both hvg and gvh nonpermissive mismatches lead to alloreactivity in the gvh direction ( i.e. , gvhd ) [ 4 , 91 ] .
therefore , both hvg and gvh nonpermissive mismatches are considered overall nonpermissive [ 4 , 91 ] .
the in silico model histocheck has been developed to estimate t - cell alloreactivity between hla class - i and class - ii mismatches .
histocheck calculates a matching score for any donor - recipient combination based on their hla typing , the so - called sequence - similarity matching score .
the sequence - similarity matching score is determined by comparing differences in amino acids between hla alleles with regard to their functional similarity and their location in the hla molecule ; amino - acid positions involved in tcr recognition and hla - peptide binding are implemented in the sequence - similarity matching score . as a high sequence - similarity matching score represents a high level of dissimilarity between donor and recipient , correlation of the sequence - similarity matching scores with clinical outcome was expected . however , histocheck is not indicative for transplant outcome in vivo , as sequence - similarity matching scores showed no correlation with gvhd after hsct [ 9496 ] .
the inability of histocheck to be indicative for t - cell alloreactivity may be explained by several limitations of this model : histocheck does not integrate the presence of alloreactive donor t cells nor viral history in its algorithm .
additionally , the concepts of aforementioned molecular mimicry between hla : peptide complexes and unconventional docking of tcr are not included in histocheck .
since these aspects of direct t - cell recognition are complex and not fully understood , establishment of reliable , clinically relevant tools to predict direct t - cell recognition remains challenging .
an alternative approach to predict direct t - cell alloreactivity is to analyze the impact of amino acids at certain locations within hla molecules .
several amino - acid substitutions in the peptide - binding domain of hla class - i molecules are related to an increased risk of gvhd , whereas other amino - acid substitutions are related to a diminished relapse risk .
the effect of specific amino - acid changes on alloreactivity was recently investigated in a large cohort . in this study , the impact of changes on hla class - i positions 9 , 99 , 116 , and 156 for peptide binding alteration and position 77 for killer cell immunoglobulin - like receptor binding was investigated in recipients of an allogeneic hsct with a single allelic mismatch at either the hla - a , -b , or -c locus .
particularly amino - acid changes at position 116 in hla - c were associated with an increased acute gvhd risk [ 97 , 98 ] , but also changes at position 99 for hla - c and position 9 for hla - b were associated with clinical t - cell alloreactivity . by determining the effect of specific amino acids within the hla molecule , multiple amino - acid positions have been identified that influence transplantation outcome ; this knowledge may be used for donor selection .
indirect recognition of allogeneic hla acts via presentation of peptides derived from allogeneic hla molecules . over 350 of these indirectly recognizable hla - derived peptides
t - cells recognizing these peptides likely play a role in alloreactivity ; the erection of indirectly recognizing t cells after solid - organ transplantation was strongly correlated to both acute [ 100102 ] and chronic graft failure [ 102 , 103 ] .
furthermore , the presence of circulating t - cells recognizing allogeneic hla epitopes in an indirect manner was predictive of rejection . as mentioned previously ,
indirect t - cell recognition is considered to be a slower alloreactive response than direct t - cell recognition [ 33 , 34 ] .
the proposed slower rate of indirect t - cell recognition may be related to the idea that indirectly recognizing t cells arise from the naive pool , whereas directly recognizing t cells likely evolve from the memory pool , as the latter t cells are supposedly cross - reactive . since direct recognition has received most attention historically , not many methods are available to predict indirect recognition of hla disparities ; there is no in vitro system available and only one in silico model .
we have recently developed a model for in silico prediction of indirectly recognizable hla - derived peptides , the so - called pirches model ( predicted indirectly recognizable hla epitopes ) .
indirect t - cell recognition that targets allogeneic hla depends on hla - derived peptides that differ between host and graft .
hla - derived peptides that are identical between donor and recipient should be ignored by the alloimmune system , as t - cells recognizing these peptides should have been deleted from the repertoire due to thymic selection .
thus , the hvg reaction of graft rejection after solid - organ transplantation should be evoked by donor - specific peptides , whereas gvhd after hsct should be evoked by recipient - specific peptides .
we have designated the donor - specific peptides that can be recognized by the recipient as hvg - pirches and the recipient - specific peptides that can be recognized by the donor as gvh - pirches . in order to elicit indirect t - cell recognition ,
allogeneic hla proteins need to be processed into peptides and these peptides need to be presented on shared hla . since both steps are determined by certain motifs in the protein sequences , both antigen processing and antigen presentation pathways
our pirches model uses these predictions to define permissibility of hla mismatches . for hla class - i peptide presentation ( designated as pirche -
i ) , the pirches model first determines proteasomal cleavage of all hla molecules of the donor and recipient into peptides and transport of those peptides via the transporter associated proteins ( tap ) into the endoplasmic reticulum ( er ) .
subsequently , the binding affinities of the predicted cleavage products to hla class - i alleles are predicted , as a derivative of peptide presentation by the hla class - i molecules that are shared between donor and recipient .
prediction of hla class - ii - presented epitopes ( pirche - ii ) is restricted to hla - binding affinity predictions of peptides , since ( enzymatic ) cleavage patterns have not been clearly defined yet . on the basis of their performance , we implemented netchop , netmhcpan , and netmhc - ii or netmhciipan ( reviewed in [ 106109 ] ) in our pirches model to predict the number of pirche - i and pirche - ii .
netchop is a potent predictor of proteasomal cleavage and tap transport , whereas netmhcpan predicts binding affinity to hla class i. netmhc - ii can predict peptide binding to hla class - ii alleles for which binding data exist , whereas for netmhciipan these data were extrapolated to other alleles .
both hla class - i- and hla class - ii - binding predictors have good predictive capacities   and are frequently used to identify viral epitopes .
the first construction of the pirches model was based on predicting hla class - i - derived peptide presentation on shared hla - dr and used the binding affinity predictions of netmhc - ii .
after kidney transplantation with hla class - i mismatches , mismatches that led to allogeneic hla - specific antibody production correlated to higher numbers of hvg - pirche - ii compared to mismatches that did not led to antibody production , suggesting that indirect recognition of hla - derived epitopes was required for hla - specific igg antibody production . for hsct ,
the situation is more difficult , as alloreactivity after hsct not only involves cd4 + t - cell recognition and stimulation of b cells but clearly involves cd8 + t - cell recognition of alloantigens as well .
moreover , usage of netmhciipan was incorporated , as netmhc - ii can only predict binding to a limited number of hla - dr alleles .
indeed , after hla - mismatched hsct , high numbers of both gvh - pirche - i and -ii are correlated to clinical alloreactivity ( thus et al . , manuscripts in preparation ) . in the current pirches model ,
we regarded any difference in presentable peptides derived from donor - versus - recipient alleles as a pirche ( i.e. , only one amino - acid difference is regarded as difference ) .
the model can likely be improved when the t - cell recognition is more specifically elucidated .
it is well known that some positions of peptides are more important in tcr binding than others [ 123 , 124 ] , as amino acids that are lying deep inside the peptide binding groove of the presenting hla molecule are likely not seen by the tcr .
furthermore , polymorphisms leading to different peptide properties ( e.g. , polar versus nonpolar and hydrophobic versus hydrophilic ) may lead to more pronounced t - cell recognition .
these refinements are currently being studied for their effect on the predictive potential of the model .
hla mismatches can cause severe posttransplantation complications such as graft rejection   and gvhd . in these complications ,
the induction of both antibody production and t - cell recognition may play a role .
interestingly , permissibility of hla - mismatched combinations is highly variable ; some mismatches are poorly tolerated , whereas others are highly permissible .
although the degree of hla amino - acid sequence disparity varies largely amongst different hla mismatches depending on the allelic versus antigenic nature of the mismatch and the hla locus , the number of polymorphic amino - acid residues in itself is not predictive for the permissibility of hla - mismatched combinations , as multiple additional factors are involved . both the nature and the position of the amino - acid polymorphisms within the mismatched hla , as well as their effect on neighboring amino acids , determine the permissibility of hla - mismatched combinations .
several approaches have been developed to predict the permissibility of hla mismatches , thereby aiming to improve donor selection procedures .
the objective of all these approaches is to predict the development of the abovementioned antibody and t - cell recognition of allogeneic hla .
several well - established in vitro assays can be used to detect dsa that are related to impaired graft survival .
in addition to these assays , hlamatchmaker is a well - validated tool to identify which hla mismatches do not induce alloreactive humoral responses in transplantation recipients . although hlamatchmaker is a powerful predictor for acceptable hla mismatches in solid - organ transplantation , this tool is not suitable for predicting hla permissibility in the setting of hsct . with regard to direct t - cell recognition
, the risk for clinical alloreactivity can be estimated with the in vitro ctlp assay .
in addition to this in vitro assay , several in silico approaches aim at predicting direct recognition - based t - cell alloreactivity .
for example , the tce model can assess nonpermissive hla - dpb1 mismatches for hsct .
the relevance of the tce model has not yet been investigated in solid - organ transplantation .
practically , one should note that hla - dpb1 is rarely typed prospectively in the setting of solid - organ transplantation , as donor availability is more restricted than for hsct .
although histocheck has been developed to estimate direct recognition in silico for all hla loci , this model does not correlate to alloreactivity in vitro nor in vivo .
alternatively , several studies have identified amino - acid positions that are influencing transplantation outcome ; this information can be implemented in donor selection procedures .
this model predicts hla - derived epitopes that can be presented on shared hla classes i and ii .
both pirche - i and pirche - ii are well correlated to alloreactivity after hsct . with regard to pirche - ii ,
increasing numbers of pirche - ii are correlated to antibody production after solid - organ transplantation .
alloreactivity after transplantation can unlikely be attributed to one single pathway of hla recognition . to determine the relative contribution of direct and indirect recognition , combining the different methods of predicting alloreactivity would be of interest .
direct and indirect recognition may act synergistically , and therefore the combination of a positive ctlp assay and a high number of pirches may lead to a more pronounced alloreactive response .
furthermore , combining the pirches and the tce models for hla - dpb1 mismatches might allow identification of hla - dpb1 mismatches recognized in both direct and indirect manners .
moreover , a combination of low pirche - ii and low number of eplets as determined by hlamatchmaker may be favorable in solid - organ transplantation .
in conclusion , over the past decades , many approaches have been developed to predict alloreactivity after transplantation in vivo , some attempts leading to more successful predictors than others .
the failure of multiple tools to predict alloreactivity is not surprising , as knowledge about alloreactivity is still limited .
however , multiple approaches seem to be clinically relevant and some are currently implemented in clinical practice . further improvement of the definition of hla - mismatch permissibility , and implementation of these definitions into the donor - selection procedure , will eventually lead to reduced alloreactivity , thereby improving clinical outcome after solid - organ transplantation and hsct .
 human leukocyte antigen ( hla ) mismatching leads to severe complications after solid - organ transplantation and hematopoietic stem - cell transplantation . 
 the alloreactive responses underlying the posttransplantation complications include both direct recognition of allogeneic hla by hla - specific alloantibodies and t cells and indirect t - cell recognition . 
 however , the immunogenicity of hla mismatches is highly variable ; some hla mismatches lead to severe clinical b - cell- and t - cell - mediated alloreactivity , whereas others are well tolerated . 
 definition of the permissibility of hla mismatches prior to transplantation allows selection of donor - recipient combinations that will have a reduced chance to develop deleterious host - versus - graft responses after solid - organ transplantation and graft - versus - host responses after hematopoietic stem - cell transplantation . 
 therefore , several methods have been developed to predict permissible hla - mismatch combinations . in this review 
 we aim to give a comprehensive overview about the current knowledge regarding hla - directed alloreactivity and several developed in vitro and in silico tools that aim to predict direct and indirect alloreactivity . 