Original Article

a1,3Galactosyltransferase knockout pigsproduce the natural anti-Gal antibody andsimulate the evolutionary appearance of thisantibody in primates

Galili U. a1,3Galactosyltransferase knockout pigs produce the naturalanti-Gal antibody and simulate the evolutionary appearance of thisantibody in primates. Xenotransplantation 2013: 20: 267–276. © 2013John Wiley & Sons A/S.

Abstract: Background: Anti-Gal is the most abundant natural anti-body in humans and Old World primates (apes and Old World mon-keys). Its ligand, the a-gal epitope (Gala1-3Galb1-4GlcNAc-R), isabundant in nonprimate mammals, prosimians and New World mon-keys whereas it is absent in humans and Old World primates as a resultof inactivation of the a1,3galactosyltransferase (a1,3GT) gene in ances-tral Old World primates, as recent as 20–28 million years ago. Sinceanti-Gal has been a “forbidden” autoantibody for >140 million years ofevolution in mammals producing a-gal epitopes it was of interest todetermine whether ancestral Old World primates could produce anti-Gal once a-gal epitopes were eliminated, i.e. did they carry anti-Galencoding immunoglobulin genes, or did evolutionary selection eliminatethese genes that may be detrimental in mammals synthesizing a-gal epi-topes. This question was studied by evaluating anti-Gal prodution ina1,3GT knockout (GT-KO) pigs recently generated from wild-type pigsin which the a-gal epitope is a major self-antigen.Methods: Anti-Gal antibody activity in pig sera was assessed byELISA, flow cytometry and complement mediated cytolysis and com-pared to that in human sera.Results: The study demonstrates abundant production of the naturalanti-Gal antibody in GT-KO pigs at titers even higher than in humans.The fine specificity of GT-KO pig anti-Gal is identical to that of humananti-Gal.Conclusions: Pigs and probably other mammals producing a-gal epi-topes carry immunoglobulin genes encoding anti-Gal as an autoanti-body. Once the a-gal epitope is eliminated in GT-KO pigs, they produceanti-Gal. These findings strongly suggest that similar to GT-KO pigs,inactivation of the a1,3GT gene in ancestral Old World primatesenabled the immediate production of anti-Gal, possibly as a protectiveantibody against detrimental microbial agents carrying a-gal epitopes.

Uri GaliliDepartment of Surgery, University of MassachusettsMedical School, Worcester, MA, USA

Key words: anti-Gal – GT-KO pigs – primateevolution – a1, 3galactosyltransferase – a-galepitope

Abbreviations: a1,3GT, a1,3galactosyltransferase;a-gal epitope, the carbohydrate antigen Gala1-3Galb1-4GlcNAc-R; GT-KO, a1,3galactosyltransfer-ase gene knockout; WT, wild type.

Address reprint requests to Dr. Uri Galili,Department of Surgery, University of MassachusettsMedical School, 55 Lake Avenue North, Worcester,MA 01655, USA (E-mail: uri.galili@RCN.com)

Received 8 July 2013;Accepted 31 July 2013

Introduction

The anti-Gal-mediated barrier of hyperacute rejec-tion of pig xenografts, observed in humans andmonkeys, has been overcome with the generationof knockout pigs for the a1,3galactosyltransferasegene (GT-KO pigs) [1–4]. Whereas anti-Gal bindsto the multiple a-gal epitopes (Gala1-3Galb1-

4GlcNAc-R) expressed on cells of xenografts fromwild-type (WT) pigs and destroys them by bothcomplement-mediated cytolysis and antibody-dependent cell-mediated cytotoxicity (ADCC),GT-KO pig cells lack a-gal epitopes and thus arenot destroyed by anti-Gal [1–4]. In WT pigs andother non-primate mammals, the a-gal epitopeis synthesized within the Golgi apparatus by the

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Xenotransplantation 2013: 20: 267–276 © 2013 John Wiley & Sons A/SPrinted in Singapore. All rights reserveddoi: 10.1111/xen.12051

XENOTRANSPLANTATION

glycosylation enzyme a1,3galactosyltransferase(a1,3GT) [5–8]. The generation of these GT-KOpigs in the last decade by disruption (knockout) ofthe a1,3GT gene (also called GGTA1) [9,10] raisedthe question whether the loss of a-gal epitopes inthese pigs resulted in them gaining the ability toproduce the natural anti-Gal antibody. This ques-tion is of interest since it can shed light on the timeanti-Gal appeared in ancestral Old World monkeysand apes (collectively referred to as Old World pri-mates).

Anti-Gal is the most abundant natural antibodyin humans constituting ~1% of immunoglobulins[11,12]. Moreover, as many as 1% of human Bcells are capable of producing the anti-Gal anti-body [13]. Whereas anti-Gal is produced inhumans and Old World primates, its naturalligand, the a-gal epitope is produced in large num-bers in non-primate mammals (e.g., kangaroo,mouse, rat, rabbit, pig, dog, horse, lion, and dol-phin), prosimians (e.g., lemurs), and New Worldmonkeys (monkeys of Central and South America)[8,14–16]. Since the a-gal epitope is abundant inboth marsupials and placental mammals and sinceit is absent in non-mammalian vertebrates (fish,amphibians, reptiles, and birds), it is most proba-ble that the a1,3GT gene and its product the a-gal

epitope appeared in mammalian evolution at least140 million years ago (i.e., before marsupials andplacental mammals diverged from a commonancestor) (Fig. 1). This implies that anti-Gal hasbeen a “forbidden” autoantibody in all mammalsthat have been synthesizing the a-gal epitopethroughout > 140 million years of mammalian evo-lution. Production of this antibody in a-gal epi-tope-producing mammals is likely to result indetrimental autoimmune phenomena followinganti-Gal/a-gal epitope interaction [17]. The con-stant prevention of anti-Gal production in mam-mals by natural selection throughout thisevolutionary period might have resulted in theelimination of anti-Gal-encoding immunoglobulingenes from the mammalian gene repertoire.

The only known exceptions for anti-Gal produc-tion in mammals is Old World monkeys, apes, andhumans [15,16], all having an inactive a1,3GT geneas the result of few single-base deletions that gener-ate premature stop codons that truncate theenzyme molecule and thus inactivate it [18–24].Based on the sequence of the a1,3GT pseudogenein Old World primates and humans, it has beensuggested that the inactivation of the a1,3GT genein ancestral Old World primates occurred 20–28million years ago [18,20,23] (Fig. 1) and that it

Fig. 1. Evolutionary period in which a-gal epitope elimination and the ensuing natural anti-Gal antibody production were initiatedin primates. The synthesis of a-gal epitopes in mammals and in no other vertebrates suggests that the a1,3GT enzyme and its biosyn-thetic product the a-gal epitope appeared in ancestral mammals before marsupials and placental mammals diverged from a commonancestor (estimated 125–140 million years ago [mya]). a-Gal epitope synthesis has been conserved in lemurs (prosimians that divergedfrom primates 60–70 mya) and in New World monkeys (diverged from Old World primates 30–40 million years ago). Divergence ofOld World monkeys and apes from a common ancestor is estimated to have occurred 25–30 mya. The dashed lines mark the esti-mated evolutionary period in which ancestral Old World primates began to be subjected to the evolutionary selective pressure for theinactivation of the a1,3GT gene (based on ref. 8,18–24). The evolutionary periods of divergence in mammals are based on severalsources, in particular D. Pilbeam. “The descent of hominoids and hominids,” Sci. Am. 250: 84–96, 1984, and R. Dawkins “TheAncestor’s Tale,” Houghton Mifflin Harcourt, 2004.

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could be associated with a major catastrophic epi-demiological event which affected only ancestralOld World primates [24]. New World monkeysand lemurs (evolving in Madagascar) have notbeen subjected to this selective pressure since theyhave evolved in geographic areas that are sepa-rated from the Old World land mass by oceanicbarriers. Primates with an inactivated a1,3GT genelacked the a-gal epitope and thus were not immu-notolerant to it. The anti-Gal antibody, if pro-duced following inactivation of the a1,3GT gene,could provide immune protection to ancestral OldWorld primates against pathogens endemic to theOld World land mass that were detrimental to pri-mates that expressed a-gal epitopes [8,24].

The evolutionary inactivation of the a1,3GTgene in ancestral Old World primates 20–28 mil-lion years ago vs. the constant prevention of anti-Gal production in mammals for > 140 millionyears raises the question whether ancestral OldWorld primates had the genetic information intheir immunoglobulin gene repertoire for produc-ing anti-Gal immediately after the a1,3GT genewas inactivated. This question could be addressedin GT-KO pigs since their a1,3GT gene was inacti-vated within the last decade [9,10], similar to theinactivation of the primate a1,3GT gene 20–28 mil-lion years ago. Production in GT-KO pigs of thenatural anti-Gal antibody with characteristics simi-lar to those of the human natural anti-Gal anti-body would imply that anti-Gal immunoglobulingenes have been part of the mammalian immuno-globulin gene repertoire (including that of ances-tral primates) despite the constant evolutionarypressure against the production of this antibodyfor > 140 million years. This would strongly sug-gest the production of anti-Gal in ancestral OldWorld primates immediately followed the elimina-tion of a-gal epitopes.

In contrast, lack of the natural anti-Gal anti-body in GT-KO pigs would imply that a-galepitope-producing mammals lack the genes encod-ing anti-Gal in their immunoglobulin gene reper-toire because of the evolutionary pressure againstproduction of this antibody in the presence ofa-gal epitopes. This would further suggest thatancestral Old World primates lacked the ability toproduce anti-Gal immediately upon elimination ofthe a-gal epitope and that production of this anti-body required additional evolution of the immuno-globulin gene repertoire for the appearance ofimmunoglobulin genes encoding this antibody.Recent studies measuring anti-Gal activity by anti-body binding to synthetic a-gal epitopes linked topolyacrylamide demonstrated the production ofthis antibody in GT-KO pigs [25,26]. In addition,

binding of GT-KO pig natural antibodies to WTpig mononuclear cells and complement-mediatedcytolysis following this antibody binding [27]implied the production of anti-Gal in GT-KO pigs.

In the present study, anti-Gal in GT-KO pigswas evaluated for production at various ages,interaction with natural mammalian a-galepitopes, and for similarity in characteristics withthe human natural anti-Gal antibody. Anti-Galwas found to be produced in GT-KO-pigs at theearly age of 1.5 months and to display characteris-tics similar to those of human anti-Gal. Theseobservations imply that mammals producing a-galepitopes have carried immunoglobulin genesencoding anti-Gal. Thus, it is likely that productionof this natural antibody occurred immediately inthe first Old World primates that were homozygousfor the inactivated a1,3GT gene, similar to the pro-duction of anti-Gal observed in GT-KO pigs.

Methods

GT-KO pig sera and materials

GT-KO pigs were purchased from Fios Therapeu-tics Inc (Rochester, MN, USA) [28]. Sera of GT-KO pigs and WT pigs were obtained from FiosTherapeutics. Peroxidase (HRP)-conjugated rabbitanti-human IgG antibody was purchased fromDako (Copenhagen, Denmark), HRP-anti-pig IgGantibody and fluorescein (FITC)-labeled anti-pigIgG antibody from Sigma (St. Louis, MO, USA),HRP-anti-mouse IgM antibody from AccurateLabs (Westbury, NY, USA), FITC-labeled anti-pig IgM antibody from KPL (Gaithersburg, MD,USA), and FITC-labeled anti-human IgG andIgM antibodies from BD Pharmingen (San Diego,CA, USA). Free N-acetyllactosamine was pur-chased from Sigma, and free synthetic a-gal trisac-charide was synthesized as previously described [29]and was a generous gift from Fios Therapeutics.Synthetic a-gal epitope linked to BSA (a-gal BSA)used as solid-phase antigen for measuring anti-Galactivity in ELISA was purchased from Dextra(Reading, UK). Monoclonal anti-Gal antibodyM86 was produced in the author’s laboratory [30].

ELISA inhibition assay

Expression of a-gal epitopes on pig kidney wasquantified by the ELISA inhibition assay, an assayanalogous to a radioimmunoassay [30,31]. WT pigkidney or GT-KO pig kidney homogenates at con-centrations of 0.75–200 mg/ml were coincubatedin microfuge tubes with the monoclonal anti-GalIgM antibody M86 (at a dilution yielding

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50% maximum binding) with constant rotation for20 h at 4 °C. At the end of incubation, kidneyfragments and the immunocomplexed anti-Galwere removed by centrifugation, and the superna-tants were evaluated for the presence of monoclo-nal anti-Gal by ELISA with a-gal BSA assolid-phase antigen, using HRP-anti-mouse IgMas secondary antibody. The concentration of anti-Gal remaining in the supernatant is inversely pro-portional to the concentration of a-gal epitopes inthe tissue fragments. A high a-gal epitope concen-tration on the fragmented tissue will result in sub-sequent inhibition of anti-Gal binding to a-galBSA, even at low concentrations of pig kidneyhomogenate. Absence of a-gal epitopes wouldresult in 0% inhibition of anti-Gal binding in theELISA wells even at the highest kidney homoge-nate concentration of 200 mg/ml.

Anti-Gal activity assessed by ELISA

Anti-Gal IgG activity in pig and human sera wasassessed by ELISA with a-gal BSA as solid-phaseantigen [31]. ELISA wells were coated overnightwith 10 lg/ml a-gal BSA then blocked with 1%BSA in PBS. Sera were placed in the wells at serialtwofold dilutions for 2 h at room temperature.After washing, secondary HRP-coupled anti-pigIgG or anti-human IgG antibodies were placed inwells for 1 h followed by color development withortho-phenylene diamine (Sigma). The color reac-tion was stopped after 5 min with 1 M sulfuricacid, and light absorbance was measured in anELISA reader at 492 nm. Anti-Gal IgM binding toa-gal epitopes could not be determined by ELISAbecause of the non-specific binding of serum IgMmolecules to ELISA wells.

Flow cytometry analysis of anti-Gal activity

Evaluation of pig and human anti-Gal IgM andIgG binding to a-gal epitopes on nucleated cellswas performed using B16 mouse melanoma cellsthat completely lack a-gal epitopes and the samecells that underwent stable transfection witha1,3GT gene for the expression of multiple a-galepitopes (B16agal cells) [32]. B16 cells and B16agalcells were incubated for 1 h at 4 °C with pig orhuman sera diluted 1 : 10. Subsequently, the cellswere washed and incubated for 30 min at 4 °Cwith the corresponding FITC-coupled secondaryantibody, washed, resuspended in 2% paraformal-dehyde, and subjected to staining analysis in aFACSCalibur flow cytometer (Becton Dickinson,San Jose, CA, USA). Increase in staining ofB16agal cells vs. B16 cells indicates the specific

binding of anti-Gal to a-gal epitopes on the for-mer cells.

Results

Absence of a-gal epitopes in GT-KO pigs

Studies with pig and rat cells have indicated that inaddition to the a1,3GT gene there is a separatebiosynthetic pathway that produces low level ofa-gal epitopes [33,34]. Thus, it was of interest todetermine whether GT-KO pigs synthesize lowamounts of a-gal epitopes in their organs. Thisanalysis was performed in GT-KO pig kidneyssince in a previous study [35], expression of a-galepitopes in WT pig kidney was shown to be muchhigher than that in WT pig lung or heart, and theconcentration of this epitope in WT pig kidney is~500-fold higher than that in WT mouse kidney.Quantification of a-gal epitopes in pig kidneyhomogenates was performed by the ELISA inhibi-tion assay that measures binding of monoclonalanti-Gal M86 antibody to fragmented tissues[30,31]. WT pig kidney fragments bind effectivelymonoclonal anti-Gal even at the low tissue homog-enate concentration of ~1.0 mg/ml (Fig. 2). Thisbinding corresponds to ~1 9 1012 a-gal epitopes/mg tissue [31]. In contrast, GT-KO pig kidneyfragments bind no anti-Gal even at a ~200-fold

Fig. 2. Quantification of a-gal epitopes in kidneys from twoWT pigs (D,○) and GT-KO pigs (▲,●) by ELISA inhibitionassay. The assay measures monoclonal anti-Gal M86 bindingto homogenates of fragmented kidney tissue by evaluatinginhibition of subsequent binding of the free antibody in super-natants to a-gal BSA. The 50% inhibition of monoclonal anti-Gal binding at 1–2 mg/ml of WT kidney tissue homogenatecorresponds to expression of ~1012 a-gal epitopes/mg tissue[31]. GT-KO pig kidney fragments completely lack a-gal epi-topes, and thus, do not inhibit anti-Gal binding to a-gal BSA.

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higher concentration of the homogenate (i.e.,200 mg/ml), implying that GT-KO pigs lackdetectable a-gal epitopes (Fig. 2). This confirmsthe assumption that inactivation of the a1,3GTgene in GT-KO pigs simulates the evolutionaryinactivation of this gene in ancestral Old Worldprimates in that a-gal epitope synthesis has beeneliminated in both groups.

GT-KO pigs produce the natural anti-Gal antibody

Anti-Gal IgG production in 1-2y GT-KO pigs wasstudied by ELISA using synthetic a-gal epitopeslinked to bovine serum albumin (a-gal BSA) assolid-phase antigen (Fig. 3A). Analysis of IgGbinding to a-gal BSA demonstrated anti-Gal activ-

ity in GT-KO pig sera at titers even higher thanthose in human sera. As expected, WT pigs pro-duce no anti-Gal antibody as these pigs synthesizea-gal epitopes and are immunotolerant to it(Fig. 3A). Production of the natural anti-Gal anti-body in GT-KO pig is age dependent. It is notfound at the age of 1 week and is low at 1 month(Fig. 3B). However, by 1.5 months, anti-Gal activ-ity is close to that in older GT-KO pigs. Demon-stration of natural anti-Gal antibody productionin GT-KO pigs further supports the notion thatthese pigs completely lack a-gal epitopes (Fig. 2).If such epitopes would have been synthesized inthese pigs, no anti-Gal production would be feasi-ble as the pigs would be immunotolerant to thea-gal epitope.

Fig. 3. Anti-Gal production in pigs and humans. (A) Production of anti-Gal IgG in 1-2y GT-KO pigs (●), humans (▲), and WTpigs (○) as determined by ELISA with a-gal BSA as solid-phase antigen. HRP-rabbit anti-human IgG or HRP-anti-pig IgG servedas secondary antibodies. (B) Production of anti-Gal IgG antibody in GT-KO pigs at various ages as determined by ELISA. (C) Com-plement-mediated cytolysis of WT pig RBC incubated for 30 min at 37 °C in sera of GT-KO pigs (●) and humans (▲). GT-KO pigsera in which complement was heat-inactivated for 30 min at 56 °C are presented as (○). Rabbit serum diluted 1 : 15 was present inall serum solutions as standard complement source and was heat-inactivated in (○). The data in (A) and (C) are representative of 6pigs per group. The data in (B) represent of 2–3 GT-KO pigs at each age.

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The human natural anti-Gal antibody was foundto induce complement-mediated lysis of mamma-lian cells presenting a-gal epitopes [36–38] as wellas lysis of enveloped viruses [39,40] and protozoasuch as Trypanosoma cruzi that present a-galepitopes [41]. In view of these observations, it hasbeen assumed that anti-Gal could serve in ances-tral Old World primates as a means of defenseagainst detrimental pathogen(s) expressing a-galepitopes [8,15,24]. Thus, it was of interest to deter-mine whether anti-Gal produced in GT-KO pigscan induce complement-mediated cytolysis of cellsexpressing a-gal epitopes. This was studied withWT pig red blood cells (RBC) incubated with GT-KO pig sera. Such incubation resulted in comple-ment-mediated cytolysis at titers even higher thanthose observed with anti-Gal in human sera(Fig. 3C). Heat inactivation of complementresulted in no cytolysis. The GT-KO pig antibodymediating this cytolysis is anti-Gal as WT pig RBCand GT-KO pig RBC differ only in expression ofmultiple a-gal epitopes on the former [9,10].

Comparison of the binding of GT-KO pig anti-Gal IgM vs. IgG antibodies to a-gal epitopes on

cells could be evaluated by flow cytometry withmouse melanoma B16 cells lacking a-gal epitopesand with the same cells transfected with a1,3GTcDNA (designated B16agal cells), which express~106 a-gal epitopes per cell [32]. Binding of bothGT-KO pig anti-Gal IgG and human anti-Gal IgGto B16agal cells was higher than anti-Gal IgM bind-ing in each of the sera (Fig. 4). This suggests thatmany of the anti-Gal-producing B cells in GT-KOpigs, as well as in humans, undergo isotype switchfrom IgM into IgG production soon after leavingthe bone marrow. Similar to the results in ELISAwith a-gal BSA (Fig. 3B), 4-week-old GT-KO pigsdisplayed an initiation of anti-Gal IgG and IgMresponse, and no anti-Gal production was detectedwith WT pig sera (Fig. 4).

GT-KO pig anti-Gal and human anti-Gal display similar fine

specificity

Inhibition of binding of the natural GT-KO piganti-Gal and of human anti-Gal IgG antibodies toa-gal BSA was determined with free oligosaccha-rides to study the similarity in the fine specificity of

GT-KO pig 4 weeks

Anti-Gal IgG

Anti-Gal IgM

GT-KO pig 3 months

GT-KO pig 9 months

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Humanadult

Anti-Gal IgM

Humanadult

WT pigadult

Fig. 4. Relative binding of anti-Gal IgM and IgG in GT-KO pig, WT pig, and human sera. Sera (diluted 1 : 10) were studied foranti-Gal binding by flow cytometry with cells lacking a-gal epitopes (B16 melanoma cells - open curves) and to the same cells which,however, express ~106 a-gal epitopes/cell as a result of stable transfection with the a1,3GT gene (B16agal cells - closed curves). The is-otype control for each serum overlaps the open curve in the left figure in each row. Note that both GT-KO pig and human sera dis-played a higher anti-Gal IgG activity than the IgM isotype of this antibody.

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anti-Gal in the two species. Free a-gal epitope tri-saccharide (Gala1-3Galb1-4GlcNAc) inhibitedbinding of anti-Gal in sera of both species, whereasfree N-acetyllactosamine (Galb1-4GlcNAc), thatis, the a-gal epitope trisaccharide without theterminal a1,3galactosyl unit, failed to inhibit anti-Gal binding (Fig. 5A). Moreover, both GT-KOpig anti-Gal and human anti-Gal were found tobind to a-gal epitopes on WT pig kidney fragmentsand thus were removed from the serum followingincubation with these kidney fragments. Thus,incubation of GT-KO pig sera and of human serawith WT pig fragments results in subsequent lossof IgG binding to a-gal BSA in ELISA (Fig. 5B).As expected, anti-Gal activity in both GT-KO pigand human sera was not affected by incubationwith GT-KO pig kidney fragments as these tissuefragments lack a-gal epitopes.

Discussion

The present study demonstrates production ofthe natural anti-Gal antibody in GT-KO pigs attiters that are even higher than those measured inhumans, when evaluated by ELISA with syn-thetic a-gal epitopes linked to BSA as solid-phaseantigen. These findings confirm previous observa-tions of Yeh et al. [25] and Fong et al. [26] onthe interaction between anti-Gal produced byGT-KO pigs and synthetic a-gal epitopes. Inaddition, inhibition with free oligosaccharidesperformed in the present study indicated that thenatural anti-Gal antibody produced in GT-KOpigs displays a fine specificity that is identical tothat of the natural human anti-Gal antibody.Moreover, both GT-KO pig and human anti-Galbind to natural mammalian a-gal epitopes on

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Fig. 5. Comparison of anti-Gal fine specificity in GT-KO pig and human sera as determined by inhibition of IgG antibody bindingin the presence of free oligosaccharides (A) and by adsorption on WT pig kidney homogenate fragments (B). A. Inhibition of anti-Gal in GT-KO pig sera and human sera (diluted 1 : 50) from binding to a-gal BSA in ELISA by oligosaccharides. Open columns -serum with no oligosaccharides; closed columns - serum containing 10 mM free a-gal epitope oligosaccharide (Gala1-3Galb1-4GlcNAc); hatched columns - serum containing 10 mM N-acetyllactosamine (Galb1-4GlcNAc). Mean + SD is indicated for eachspecies. B. Binding of GT-KO pig anti-Gal and of human anti-Gal to a-gal epitopes on WT pig kidney fragments, as measured bysubsequent ELISA with a-gal BSA. Sera diluted 1 : 10 were incubated for 2 h at 4 °C with 100 mg/ml WT pig kidney homogenate(●), GT-KO pig kidney homogenate (■), or with no pig kidney homogenate (▲), with constant rotation. Subsequently, the suspen-sions were spun and the sera subjected to ELISA for determining anti-Gal IgG activity. Data in (A) and (B) are representative of 5adult GT-KO pig or human sera in each group.

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WT pig kidney fragments and on mouse B16melanoma cells transfected with the a1,3GT gene.These two natural anti-Gal antibodies furtherbind to a-gal epitopes on WT pig RBC andinduce complement-mediated hemolysis of theseRBC. A similar complement-mediated cytolysiswas previously observed with WT pig mononu-clear cells incubated in GT-KO pig sera [27]. Asexpected, no anti-Gal production has beenobserved in WT pigs as the a-gal epitope is amajor self-antigen in these pigs, and thus, theyare completely immunotolerant to it. The onlyknown difference between anti-Gal in humansand in GT-KO pigs is that in humans the mater-nal anti-Gal IgG crosses the placenta and isfound in the cord blood [11]. In contrast, noanti-Gal IgG antibodies were detected in 1-week-old GT-KO pig newborns, as the six-layeredstructure of the pig placenta prevents passage ofmaternal immunoglobulins into the fetal circula-tion [42].

The observed production of the natural anti-Galantibody in GT-KO pigs implies that WT pigs andtheir ancestors have been carrying anti-Gal-encod-ing genes in their immunoglobulin gene repertoiredespite the potential risk of anti-Gal acting as a“forbidden” autoantibody throughout mammalianevolution. If such immunoglobulin genes wouldhave been absent from the immunoglobulin generepertoire due to an evolutionary selection process,it is highly unlikely that they could evolve in theshort period of the few years since the GT-KOpigs were generated [9,10]. This finding stronglysuggests that the immunoglobulin gene repertoirecontains the information for production of detri-mental autoantibodies and that the actual produc-tion of such antibodies is prevented only by theimmune tolerance mechanism toward self-a-galepitopes.

This observation on anti-Gal-encoding immuno-globulin genes in non-primate mammals is furthersupported by previous studies in GT-KO mice.These knockout mice were generated in the mid-1990s by disruption of the a1,3GT gene [43,44].Although these mice do not naturally produceanti-Gal, they produce this antibody after immuni-zation with xenogeneic cells expressing a-gal epi-topes [32,45,46]. WT mice do not produce anti-Gal, even after such immunizations as the a-galepitope is a self-antigen in WT mice, as it is in WTpigs. It is possible that the sterile environment andsterile food provided to GT-KO mice prevent thedevelopment of gastrointestinal flora which inhumans mediates the continuous antigenic stimula-tion for anti-Gal production [47]. Anti-Gal inGT-KO mice was found to be encoded by

immunoglobulin genes VH22.1, VH441, andVK5.1, all found also in WT mice [48,49]. Interest-ingly, anti-Gal in humans is encoded by severalheavy chain genes primarily of the VH3 immuno-globulin gene family [50,51].

The presence of immunoglobulin genes encodinganti-Gal in WT pigs and WT mice implies thatthese genes have been present also in other mam-mals which synthesize a-gal epitopes. The presenceof these genes in the ancestral primate immuno-globulin gene repertoire was probably of cardinalsignificance in the evolution of Old World primatesby enabling the protection of these primates frompathogens presenting a-gal epitopes. There aremany microbial pathogens known to present a-galepitopes, including enveloped viruses [39,40,52,53],bacteria such as Klebsiella, Salmonella, Streptococ-cus, and E. coli [47,54,55], and protozoa such asTrypanosoma and Leishmania [41,56–58]. Thesemultiple examples of a-gal-presenting pathogensraise the possibility that the selective pressure forinactivation of the a1,3GT gene in primates, whichwas endemic for the African, Asian, and Europeanland mass (the Old World), was mediated by adetrimental microbial pathogen presenting a-galepitopes. Prior to this selective pressure, ancestralOld World primates lacked the ability to mount aneffective immune protection against such patho-gens by producing protective anti-Gal antibody,similar to the inability of WT pigs or WT mice toproduce anti-Gal. However, once the mating ofancestral Old World monkeys and apes heterozy-gous for the inactivated a1,3GT gene gave rise tooffspring that were homozygous for the inactivatedgene, production of the natural anti-Gal antibodybecame feasible in the absence of a-gal epitopes, asin GT-KO pigs. The de novo generated anti-Galantibody could protect these ancestral Old Worldprimates lacking a-gal epitopes against a-gal-expressing pathogens and provided a significantsurvival advantage over primates synthesizing thea-gal epitope.

In addition to the use of GT-KO pig organs andtissues for avoiding anti-Gal-mediated hyperacuterejection, production of the natural anti-Gal anti-body in these pigs also makes these an excellentrecipient candidate for studying anti-Gal-mediatedrejection of a pig xenograft without any interfer-ence of other anti-xenograft mechanisms, such asanti-non-Gal antibody or a T-cell response.

Acknowledgment

The author thanks Drs. J. Logan and B. Weissmanof Fios Therapeutics Inc. for sera of GT-KO pigsof different age groups.

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Disclosure

The author declares no conflict of interests andother financial interests in this study.

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