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 .