Does human leukocyte antigens sensitization matter for xenotransplantation?

The major histocompatibility complex class I and class II human leukocyte antigens (HLA) play a central role in adaptive immunity but are also the dominant polymorphic proteins targeted in allograft rejection. Sensitized patients with high levels of panel‐reactive anti‐HLA antibody (PRA) are at risk of early allograft injury, rejection, reduced allograft survival and often experience prolonged waiting times prior to transplantation. Xenotransplantation, using genetically modified porcine organs, offers a unique source of donor organs for these highly sensitized patients if the anti‐HLA antibody, which places the allograft at risk, does not also enhance anti‐pig antibody reactivity responsible for xenograft rejection. Recent improvements in xenotransplantation efficacy have occurred due to improved immune suppression, identification of additional xenogeneic glycans, and continued improvements in donor pig genetic modification. Genetically engineered pig cells, devoid of the known xenogeneic glycans, minimize human antibody reactivity in 90% of human serum samples. For waitlisted patients, early comparisons of patient PRA and anti‐pig antibody reactivity found no correlation suggesting that patients with high PRA levels were not at increased risk of xenograft rejection. Subsequent studies have found that some, but not all, highly sensitized patients express anti‐HLA class I antibody which cross‐reacts with swine leukocyte antigen (SLA) class I proteins. Recent detailed antigen‐specific analysis suggests that porcine‐specific anti‐SLA antibody from sensitized patients binds cross‐reactive groups present in a limited subset of HLA antigens. This suggests that using modern genetic methods, a program to eliminate specific SLA alleles through donor genetic engineering or stringent donor selection is possible to minimize recipient antibody reactivity even for highly sensitized individuals.

duced allograft survival and often experience prolonged waiting times prior to transplantation. Xenotransplantation, using genetically modified porcine organs, offers a unique source of donor organs for these highly sensitized patients if the anti-HLA antibody, which places the allograft at risk, does not also enhance anti-pig antibody reactivity responsible for xenograft rejection. Recent improvements in xenotransplantation efficacy have occurred due to improved immune suppression, identification of additional xenogeneic glycans, and continued improvements in donor pig genetic modification. Genetically engineered pig cells, devoid of the known xenogeneic glycans, minimize human antibody reactivity in 90% of human serum samples.
For waitlisted patients, early comparisons of patient PRA and anti-pig antibody reactivity found no correlation suggesting that patients with high PRA levels were not at increased risk of xenograft rejection. Subsequent studies have found that some, but not all, highly sensitized patients express anti-HLA class I antibody which cross-reacts with swine leukocyte antigen (SLA) class I proteins. Recent detailed antigen-specific analysis suggests that porcine-specific anti-SLA antibody from sensitized patients binds cross-reactive groups present in a limited subset of HLA antigens. This suggests that using modern genetic methods, a program to eliminate specific SLA alleles through donor genetic engineering or stringent donor selection is possible to minimize recipient antibody reactivity even for highly sensitized individuals.

K E Y W O R D S
anti-HLA, non-Gal antibody, panel-reactive antibody, xenoreactive antibody, interest in moving toward clinical xenotransplantation. In addition to increasing the overall supply of organs for transplantation, successful clinical xenotransplantation may be particularly helpful to sensitized patients if increased antibody reactivity to human HLA antigens does not also increase antibody reactivity to porcine donor organs. This review summarizes the literature which has examined the potential of anti-HLA antibody in allo-sensitized patients to cross-react with porcine cells. The body of evidence from these studies suggests that, at the current level of sensitivity, most transplant patients and patients with moderate allo-sensitization show minimal human antibody reactivity to pig cells when these cells lack the three known xenogeneic antigens galactose a 1,3 galactose (aGal), N-glycolylneuraminic acid (Neu5Gc) modified glycans and porcine B4GALNT2-dependent SDa glycans. 16,17 For highly sensitized patients, there is often, but not always, an increase in antipig antibody reactivity which could affect xenotransplant survival.
Recent analysis suggests that stringent patient cross-matching and/ or elimination of a limited set of specific porcine class I swine leukocyte antigens (SLA) alleles can further minimize anti-pig reactivity such that future clinical xenotransplantation may be appropriate even for highly sensitized patients.

| DE TEC TING HL A S EN S ITIZ ATI ON AND ALLOTR ANS PL ANTATION
Early clinical transplantation programs screened donor and recipients for matching blood type but did not routinely screen for evidence of sensitization to other donor antigens. In a landmark study, 18 Patel and Terasaki demonstrated that a complement-dependent cy-

| S TR ATEG IE S FOR DE TEC TING HL A CROSS-RE AC TIVIT Y FOR XENOTR ANS PL ANTATION
Xenograft rejection is recognized as an overwhelmingly antibody driven process due to the very high level of anti-pig antibodies naturally present in human serum. The bulk of these antibodies are not directed to swine SLA but bind to the major xenogeneic glycan galactose alpha 1,3 galactose (αGal). 23 With the advent of pigs engineered with a GGTA-1 mutation, 24,25 which eliminates αGal expression (GTKO), the impact of other antibodies directed to non-Gal antigen including SLA-I has become more apparent. 16,[26][27][28] There have been a limited number of studies designed to determine whether sensitization to HLA results in enhanced antibody reactivity to pig cells. 16,[29][30][31][32][33][34][35][36][37] These studies have been performed over a 20-year period and as such span a range of technological developments both for defining allo-sensitization and in technologies to detect xenoreactive antibody. In this review, the studies are presented as four basic research strategies based on the use of whole serum (type I), anti-Gal depleted serum (Type II) and anti-Gal and anti-Class I depleted serum (type III). The fourth study type largely used whole human serum but measured patient antibody reactivity to genetically modified porcine cells lacking the three known xenogeneic glycans (αGal, Neu5Gc modified glycans and SDa) with and without deletion of SLA-I genes.

| Type I studies
The earliest study 29  There was a clear reduction in antibody reactivity and CDC to GTKO cells, consistent with the loss of the αGal antigen, but no correlation between antibody reactivity or cytotoxicity to PRA level from 88 waitlisted patient sera for either WT or GTKO cell type. Cytotoxicity to porcine GTKO cells was mainly mediated by IgM antibody in contrast to anti-human cytotoxicity which was predominantly IgG dependent. Similar results were found analyzing WT and GTKO pig cells from animals with a commercial agricultural background (Large/ White, Landrace, Duroc). 36 A recent study using cells from GTKO and GTKO/CMAHKO pigs expressing human CD46 also failed to find enhanced antibody reactivity in 10 highly sensitized wait listed serum samples. 37 Collectively these studies concluded that patients with high PRA sera do not necessarily produce correspondingly high titer of anti-pig antibody or a high levels of anti-pig cytotoxicity.
Thus, allo-sensitized patients would not be at greater risk of xenograft rejection.

| Type II and III studies
Type II studies used porcine red blood cells (RBCs), which do not express SLA-I, to deplete patient serum of anti-Gal antibody and type III studies used a combination of porcine RBCs and porcine plate-

| Type IV studies
The most recent studies are based on a series of genetically modi- These studies confirm earlier reports that some, but not all, highly sensitized patient serum contains SLA-I-reactive antibody.
Importantly, these latest studies identify for the first time the MHC cross-reactive groups present on swine SLA-I making possible further genetic modification or selection to eliminate these alleles and minimize antibody reactivity even for highly sensitized patients.

| CON CLUS IONS
The prospects for clinical xenotransplantation have improved significantly due to recent increases in preclinical nonhuman primate xenograft survival. While the ideal donor organ is not universally defined and may be different for different organs, it seems likely that donor organs with minimal antigenicity (GGTA1/CMAH/ B4GALNT2) which minimize human antibody reactivity will make a prominent contribution. Additional genetic modifications to regulate complement and coagulation may also be used, but inclusion of these human transgenes should not affect antibody reactivity or tissue antigenicity. For most human sera and waitlisted patients with zero to moderate HLA sensitization, there appears to be minimal antibody reactivity to these triple knockout pig cells suggesting that future clinical xenotransplantation will be broadly applicable to most patients. Highly sensitized patients (PRA >80%) can produce antibody which cross-reacts with swine SLA-I, but this is not an obligate condition. Whether the SLA-I cross-reactive antibody in highly sensitized patients has immediate impact on xenograft survival will depend on the antibody titer, affinity and level of porcine SLA-I expression. Since recent studies suggest that cross-reactive anti-HLA antibody is directed to a limited set of HLA antigens, modern genetic screening and modification methods may be used to select for pigs with minimal antibody reactivity even for highly sensitized patients.
Patient cross-matching is a corner stone of successful allotransplantation and will undoubtedly play no less of a role in future clinical xenotransplantation.