Although HLA transgenic mice (HLA TgM) could provide a powerful approach to investigate human MHC-specific T cell responsiveness, the extent to which these molecules are recognized by the mouse immune system remains unclear. We established TgM expressing HLA class I alleles A2, B7, or B27 in their fully native form (HLAnat) or as hybrid molecules (HLAhyb) of the HLA α1/α2 domains linked to the H-2Kb α3, transmembrane, and cytoplasmic domains (i.e., to maintain possible species-specific interactions). Comparison of each as xeno- (i.e., by non-TgM) vs allo- (i.e., by TgM carrying an alternate HLA allele) transplantation Ags revealed the following: 1) Although HLAhyb molecules induced stronger xeno-CD8+ T cell responses in vitro, additional effector mechanisms must be active in vivo because HLAnat skin grafts were rejected faster by non-TgM; 2) gene knockout recipients showed that xenorejection of HLAnat and, unexpectedly, HLAhyb grafts doesn’t depend on CD8+ or CD4+ T cells or B cells; 3) each HLAhyb strain developed tolerance to “self” but rejected allele- (-B27 vs -B7) and locus- (-B vs -A) mismatched grafts, the former requiring CD8+ T cells, the latter by CD8+ T cell-independent mechanisms. The finding that recognition of xeno-HLAhyb does not require CD8+ T cells while recognition of the identical molecule in a strictly allo context does, demonstrates an α1/α2 domain-dependent difference in effector mechanism(s). Furthermore, the CD8+ T cell-independence of locus-mismatched rejection suggests the degree of similarity between self and non-self α1/α2 determines the effector mechanism(s) activated. The HLA Tg model provides a unique approach to characterize these mechanisms and develop tolerance protocols in the context of human transplantation Ags.

The MHC class I and II molecules are highly polymorphic cell surface glycoproteins centrally involved in development and responsiveness of the immune system through their capacity to present self and foreign Ag-derived peptides to T lymphocytes (1, 2). MHC class I molecules (HLA-A, -B, and -C in humans; H-2K, -D, and -L in mice) present peptides to CD8+ (cytotoxic) T cells, whereas MHC class II molecules (HLA-DR, -DP, and -DQ in humans; I-A and I-E in mice) present peptides to CD4+ (helper) T cells (1, 3, 4). Class I molecules are expressed in association with a smaller nonpolymorphic chain called β2-microglobulin (β2m)3 on most somatic cells at levels that vary significantly between tissues and cell types (5, 6). Class II molecules are found mainly on B cells, macrophages, and other APCs as well as on thymic stromal cells.

Although conventional T cell recognition of foreign (i.e., viral) Ag is self-MHC- restricted (7), CD8+ and CD4+ T cells also respond vigorously when confronted with allogeneic cells expressing non-self class I and II molecules, respectively. This potent cell-mediated immune response is believed to be the primary event involved in allograft rejection, and results from the high frequency of T cells that are alloreactive (i.e., 1–10% of the T cell repertoire) (8, 9). Although it was long believed that such cells directly recognized polymorphic differences between MHC alleles, now known to reside largely within the Ag-binding cleft (10, 11, 12), more recent evidence also suggests an important role for indirect recognition of donor MHC (or non-MHC) Ag-derived peptides in association with recipient MHC (13, 14, 15). In addition, the finding that different MHC alleles have different peptide-binding specificities (16) suggests that some alloreactive T cells may be influenced by the distinct array of self-peptides presented by foreign MHC molecules at the surface of transplanted tissues (9). Although both the direct and indirect pathways are now generally believed to be important, the relative contribution of each to allograft rejection in vivo is not clear.

In contrast to allografts, the role of the T cell response in rejection of grafts between species (i.e., xenografts) is much less characterized, in part because it occurs only after two earlier stages of rejection, namely, hyperacute rejection and delayed acute vascular rejection (17, 18, 19, 20, 21, 22). Studies conducted in vitro circumvent these earlier events and have generally detected a very low T cell response to xenogeneic cells (21, 23). This could be interpreted as resulting from either a low frequency of T cells bearing TCRs able to interact with the polymorphic domains of xenogeneic MHC and/or inefficient cross-species interactions involving coreceptor, costimulatory or accessory molecules, or other incompatibilities between species that influence T cell responsiveness. Given the multiple molecular interactions necessary for T cell activation to foreign Ag, it has been difficult to distinguish which of these is correct. This is important to resolve because, although progress has been made toward eliminating the hyperacute response (22), successful in vivo xenotransplantation protocols will require overcoming all three barriers.

Most investigations of the mechanisms of allograft rejection have been conducted with mouse strains differing at multiple major (class I and II) and/or minor MHC loci. With the exception of the nonallelic (i.e., mutant) class I (e.g., Kbm1) or class II molecules (24) and a limited number of H-2 transgenes (25, 26), few studies have addressed the influence of individual natural class I or II alleles on their own to mediate rejection. It is also unclear whether individual mouse or human alleles are functionally equivalent as transplantation Ags or whether a hierarchy exists. There are also a number of questions that need to be clarified about the cellular response to xenografts, including how complex the xenoreactive T cell population is and whether anti-xeno-MHC T cells respond to the same types of allelic polymorphisms seen by alloreactive T cells or whether there are other species-specific nonallelic differences that dominate. It will also be important to determine whether xeno-MHC molecules induce additional non-T cell effector mechanisms that contribute to rejection.

With these issues in mind, and because current mouse models are limited in what they can reveal about T cell recognition in the context of the human MHC molecules, we and others have explored the possibility that HLA class I and II molecules expressed in transgenic (Tg) mice might provide a useful model for studying HLA-dependent immune function in vivo (5, 27, 28, 29, 30, 31, 32, 33). For instance, characterization of the non-Tg mouse T cell response to human MHC expressed in tissues of otherwise genetically identical HLA Tg mice (TgM) should make it possible to identify the specific immune mechanisms involved in xeno-MHC recognition and rejection as well as the importance of MHC-dependent vs -independent interactions in the apparent reduced xenogenic cellular response detected in vitro. Furthermore, comparison of the non-Tg (i.e., xeno-) and HLA Tg (i.e., allo-) T cell responses to alternate (non-self) HLA Tg alleles could provide a unique approach to compare the structures recognized by T cells that respond to xeno- vs allo-MHC. However, despite some efforts in these directions, the extent to which Tg HLA molecules are functionally recognized by non-TgM or TgM T cells remains relatively unclear. Some reports suggest that fully human (native) HLA class I molecules are recognized only inefficiently at best by the mouse immune system as either xeno- or alloantigens, or as restriction elements (30, 31, 34, 35, 36, 37, 38, 39). In contrast, other reports, often using apparently similar alleles and strains of mice, suggest that Tg HLA class I molecules are recognized by the mouse immune system and T cells much the same as alternate mouse class I alleles (28, 29, 32, 33, 40, 41, 42, 43, 44, 45, 46, 47, 48).

It is unclear whether these inconsistencies are actually due to differential function of distinct HLA alleles in the mouse background, as opposed to other quantitative or qualitative aspects of expression, or possibly differences in specific functional assays. To distinguish between these possibilities, with the longer-term objective being development of this model as an accurate reflection of HLA function in humans, we have established a panel of TgM that express the class I alleles HLA-A2, -B7, or -B27. One set of mice expresses the fully human native heavy chain in conjunction with human β2m (hβ2m), whereas the other set expresses a hybrid form of each allele, consisting of the exons encoding the human α1 and α2 polymorphic domains (i.e., the peptide binding cleft) linked to the α3, transmembrane, and cytoplasmic domains of the mouse H-2Kb protein. Development of the HLA/H-2Kb hybrid TgM was based on results of our own and others, which suggested the possibility that species-specific molecular interactions may influence how efficiently a human class I molecule expressed in Tg cells is able to undergo intracellular interactions (i.e., with β2m, chaperones) and transport, have access to a suitable array of self and foreign peptides, or interact with the T cell coreceptor CD8 at the cell surface (6, 27, 30, 36, 37, 49, 50). As at least some of these interactions are known to depend partly or completely on class I domains outside the Ag-binding α1/α2 domains (i.e., β2m-α3; CD8-α3), the rationale for the HLA/H-2 hybrid construct was that the encoded molecule should retain the peptide-binding specificity of the human allele and be able to efficiently undergo these other interactions when expressed in a mouse background.

The studies in this paper investigate the immune mechanisms activated in vitro and in vivo by non-TgM and TgM in response to these three well-characterized human MHC class I alleles expressed as fully native HLA (HLAnat) vs HLA/H-2 hybrid (HLAhyb) Tg xeno- or allotransplantation Ags. The results demonstrate that distinct effector mechanisms are involved in recognition of, first, the identical HLA allele as a xeno- vs an allo-MHC molecule, and second, different locus-matched alleles (HLA-B7 vs -B27) vs locus-mismatched products (HLA-B vs HLA-A).

(B6/SJL)F1 (H-2b/s), C57BL/6J (BL/6) (H-2b), DBA/2J (H-2d), B6.CH-2bm1 (H-2Kbm1) (24), and IgM−/− (H-2b) (51) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). CD8−/− (52), CD4−/− (53), and CD4−/−CD8−/− (54) mice were obtained from Dr. Tak Mak (Amgen Institute and Ontario Cancer Institute, Toronto, Ontario, Canada).

The DNA constructs used to generate the HLA TgM are shown in Fig. 1. For each construct, multiple founder mice were generated by microinjection of (B6/SJL)F2 embryos (5, 55, 56), bred with non-Tg C57BL/6J mates to establish lines, and characterized with respect to Tg expression by RNA blot hybridization of tissue RNAs and by flow cytometry (5, 56). The HLA Tg lines used for these studies were selected on the basis of a normal breeding and transgene transmission rate, an appropriate tissue distribution of Tg HLA RNA (5, 56), and cell surface expression at a level similar to each other and to endogenous H-2 class I (5, 39). All constructs were genomic clones containing MHC class I exons, introns, and 5′ and 3′ flanking DNA in the genomic configuration. Each construct contained several hundred base pairs of HLA gene 5′ flanking DNA, which we and others have shown previously to include all MHC class I cis-active transcriptional regulatory information sufficient to direct appropriate HLA class I Tg expression in TgM (55, 56). The HLA-B7native (B7nat) mice carried a 6.0-kb EcoRI-BamHI fragment encoding the fully human HLA-B7 molecule (5). The HLA-B7nat/hβ2m (B7nat/hβ2m) double TgM were derived by breeding the B7nat mice above with TgM carrying a 14-kb PvuI-SaII fragment encoding hβ2m as described (5). The HLA-B27nat/hβ2m (B27nat/hβ2m) and HLA-A2nat/hβ2m (A2nat/hβ2m) mice were derived by coinjecting the human β2m gene together with either a 6.5-kb EcoRI fragment encoding HLA-B27 (57) or a 6-kb EcoRI fragment encoding HLA-A2 (58). For both the B27nat/hβ2m and A2nat/hβ2m Tg lines used here, the hβ2m and HLA genes were cotransmitted to offspring, indicating that both genes were cointegrated at a single chromosomal site.

FIGURE 1.

Schematic representation of HLA class I native (HLAnat) and hybrid (HLAhyb) transgene constructs. The HLAnat class I -A2, -B7, and -B27 TgM were generated by pronuclear microinjection of (B6/SJL)F2 embryos with DNA constructs as represented in A encoding HLA-A2, -B7, or -B27, respectively. Each construct was fully human containing all exons, introns, 5′ and 3′ flanking DNA, and 5′ transcriptional control sequences in their native genomic configuration. Aside from their natural allelic differences, the three constructs differed from each other only slightly in the amount of 5′ and/or 3′ flanking DNA. The HLA-A2nat construct was a 6.0-kb EcoRI fragment, whereas the -B7nat construct was a 6.0-kb EcoRI-BamHI fragment and the -B27nat construct was a 6.5-kb EcoRI fragment. Each of these HLAnat fragments was introduced into TgM along with a cloned genomic fragment encoding hβ2m as described (see Materials and Methods for details) (5 ). The HLAhyb class I -A2/H-2Kb (or -A2hyb), -B7/H-2Kb (or -B7hyb), and -B27/H-2Kb (or -B27hyb) TgM were generated by pronuclear microinjection of (B6/SJL)F2 embryos with DNA constructs, as represented in B, encoding the hybrid proteins HLA-A2/H-2Kb (A2hyb), -B7/H-2Kb (B7hyb), or -B27/H-2Kb (B27hyb), respectively. The A2hyb construct contained HLA-A2 5′ flanking DNA and -A2 exons 1–3 on a 2.68-kb fragment linked in intron 3 to a 2.35-kb fragment containing exons 4–8 and 3′ flanking DNA of the self H-2Kb gene (the approximate position of the HLA/H-2Kb junction is indicated by a small vertical arrowhead). The B7hyb construct contained HLA-B7 5′ flanking DNA and B7 exons 1–3 on a 1.9-kb fragment linked in intron 3 to the 2.35-kb exon 4–8 H-2Kb fragment. The B27hyb construct contained HLA-B27 5′ flanking DNA and B27 exons 1–3 on a 1.7-kb fragment linked in intron 3 to the 2.35-kb exon 4–8 H-2Kb fragment. For both the native (A) and hybrid (B) constructs, the human exons are depicted as ▨, the mouse H-2Kb exons as ▪, and 5′ and 3′ flanking DNA and introns as a thin line. The exons 1–8 are numbered below the map together with the corresponding protein domains (α1, α2, α3, transmembrane (Tm), and cytoplasmic (Cyt)). The scale is approximate.

FIGURE 1.

Schematic representation of HLA class I native (HLAnat) and hybrid (HLAhyb) transgene constructs. The HLAnat class I -A2, -B7, and -B27 TgM were generated by pronuclear microinjection of (B6/SJL)F2 embryos with DNA constructs as represented in A encoding HLA-A2, -B7, or -B27, respectively. Each construct was fully human containing all exons, introns, 5′ and 3′ flanking DNA, and 5′ transcriptional control sequences in their native genomic configuration. Aside from their natural allelic differences, the three constructs differed from each other only slightly in the amount of 5′ and/or 3′ flanking DNA. The HLA-A2nat construct was a 6.0-kb EcoRI fragment, whereas the -B7nat construct was a 6.0-kb EcoRI-BamHI fragment and the -B27nat construct was a 6.5-kb EcoRI fragment. Each of these HLAnat fragments was introduced into TgM along with a cloned genomic fragment encoding hβ2m as described (see Materials and Methods for details) (5 ). The HLAhyb class I -A2/H-2Kb (or -A2hyb), -B7/H-2Kb (or -B7hyb), and -B27/H-2Kb (or -B27hyb) TgM were generated by pronuclear microinjection of (B6/SJL)F2 embryos with DNA constructs, as represented in B, encoding the hybrid proteins HLA-A2/H-2Kb (A2hyb), -B7/H-2Kb (B7hyb), or -B27/H-2Kb (B27hyb), respectively. The A2hyb construct contained HLA-A2 5′ flanking DNA and -A2 exons 1–3 on a 2.68-kb fragment linked in intron 3 to a 2.35-kb fragment containing exons 4–8 and 3′ flanking DNA of the self H-2Kb gene (the approximate position of the HLA/H-2Kb junction is indicated by a small vertical arrowhead). The B7hyb construct contained HLA-B7 5′ flanking DNA and B7 exons 1–3 on a 1.9-kb fragment linked in intron 3 to the 2.35-kb exon 4–8 H-2Kb fragment. The B27hyb construct contained HLA-B27 5′ flanking DNA and B27 exons 1–3 on a 1.7-kb fragment linked in intron 3 to the 2.35-kb exon 4–8 H-2Kb fragment. For both the native (A) and hybrid (B) constructs, the human exons are depicted as ▨, the mouse H-2Kb exons as ▪, and 5′ and 3′ flanking DNA and introns as a thin line. The exons 1–8 are numbered below the map together with the corresponding protein domains (α1, α2, α3, transmembrane (Tm), and cytoplasmic (Cyt)). The scale is approximate.

Close modal

The hybrid HLA-B7/H-2Kb (B7/Kb or B7hyb), HLA-B27/H-2Kb (B27/Kb or B27hyb), and HLA-A2/H-2Kb (A2/Kb or A2hyb) TgM were generated by microinjection of a hybrid genomic clone containing the human exons for the α1 and α2 domains of HLA-B7, -B27, or -A2, respectively, and the mouse exons for the α3, transmembrane, and cytoplasmic domains of H-2Kb. Each hybrid construct was made by linking a 2.35-kb 3′ fragment of the genomic H-2Kb gene (beginning at a midpoint in intron 3 and containing exons 4–8 and 3′ flanking DNA) to a 5′ fragment of the specific HLA gene containing several hundred base pairs of 5′ flanking DNA and exons 1–3 up to a midpoint in intron 3.

All Tg strains were maintained by backcrossing to C57BL/6J (H-2b) mates. The strains used in the experiments described here had been backcrossed at least 8–10 generations. The HLA-B7hyb/CD8−/− and HLA-B27hyb/CD8−/− TgM were generated by breeding the corresponding HLAhyb+/−/CD8+/+ line (-B7hyb or -B27hyb) with CD8α−/− (H-2b) (52) mice and subsequently breeding the HLAhyb+/−/CD8α+/− heterozygous offspring with CD8α−/− mice. All inbred, KO, and Tg mice were housed in a pathogen-free animal facility at The Hospital For Sick Children in accordance with the current regulations and standards of the Canadian Council of Animal Care.

The DBA/2 (H-2d)-derived mastocytoma cell line P815 (59) was cotransfected by electroporation of the bacterial neomycin (G418) resistance gene pHuβpr-Neo (60) with individual HLAnat or HLAhyb gene constructs using a Bio-Rad gene pulser (Bio-Rad, Richmond, CA). Electroporated cells were cultured and selected in αMEM supplemented with 5% newborn calf serum (Sigma, St. Louis, MO) and 200 μg/ml G418 (Life Technologies, Rockville, MD) at 37°C and 5% CO2. Clones expressing high comparable levels of cell surface HLA for each transfected population were isolated by limiting dilution and characterized by flow cytometry.

The mAbs used, their specificities, and their sources were as follows: ME-1 (specific for HLA-B7, -B27, and -Bw22) was obtained from American Type Culture Collection (ATCC; Manassas, VA) (5, 61); MA2.1 (specific for HLA-A2 and -B17) was purchased from ATCC (62); B9.12.1 (pan-HLA class I-specific) was a generous gift of F. Lemmonier (Pasteur Institute, Paris, France; Ref. 63); and 28-14-8S (specific for Db, Ld, and Dq) (64) was obtained from ATCC. For flow cytometry, single cell suspensions from lymph nodes were prepared, and 1 × 106 cells were stained with the primary mAb for 45 min at 4°C in 75 μl of staining buffer (1× PBS containing 5% BSA and 0.1% sodium azide). Then, cells were washed and incubated for 30 min with FITC-conjugated F(ab′)2 goat anti-mouse IgG (Fc-specific) (Accurate Chemical and Scientific, Westbury, NY). Cells were fixed in 1% paraformaldehyde in PBS containing 0.1% sodium azide and then analyzed on a Becton Dickinson FACScan flow cytometer (Mountain View, CA).

CML assays were performed essentially as described (65). Mouse responder lymph node cells (LNCs) (1 × 104 to 3 × 105 cells/well) were cultured in 96-well round-bottom microtiter plates (Nunc, Naperville, IL) with 3 × 105 irradiated (2000 rad) stimulator cells for 5 days at 37°C and 5% CO2 in αMEM supplemented with 10% FCS (Sigma), 10 mM HEPES, 5 × 10−6 M 2-ME, and penicillin/streptomycin (Life Technologies). Stimulator cells were obtained from the spleens of (B6/SJL)F1 (H-2bs), C57BL/6 (H-2b), H-2Kbm1, and DBA/2J (H-2d) mice or HLAnat or HLAhyb class I TgM. Targets were either spleen cells that had been stimulated for 3 days with Con A (see below) or HLA-transfected P815 cells. After 5 days of in vitro stimulation, 100 μl of the culture supernatant was removed from each well and 51Cr-labeled targets (3 × 103) were added to the effector cells at the ratios indicated in a 200-μl final volume for 4 h at 37°C. Subsequently, 100 μl of supernatant was removed from each well and counted on a Wallac gamma counter (Gaithersburg, MD). Specific lysis was calculated as [(experimental − spontaneous release)/(maximal − spontaneous release)] × 100%. Spontaneous release was estimated by adding 3 × 10351Cr-labeled target cells to 100 μl of media containing stimulator but not responder cells. Maximal release was estimated by adding 3 × 10351Cr-labeled target cells to 100 μl of media containing 1% acetic acid.

Con A-stimulated lymphoblasts were generated by incubating 1 × 107 spleen-derived cells at 37°C, 5% CO2 for 2–3 days in a vertical T-25 flask in 10 ml of αMEM containing 10% FCS (Sigma), 10 mM HEPES, 5 × 10−6 M 2-ME, penicillin/streptomycin, and 2 mg/ml Con A (generously provided by Dr. R. Miller, Ontario Cancer Institute). HLA-transfected P815 cells were cultured in T-25 Falcon flasks (Becton Dickinson, Franklin Lakes, NJ) at 37°C and 5% CO2 in αMEM containing 5% newborn calf serum (Life Technologies) and penicillin/streptomycin. Cells were passed over Lympholyte-M (Cedarlane, Hornby, Ontario, Canada), washed three times, resuspended in 10 ml of medium, and counted. Con A lymphoblasts (1 × 106) or HLA-transfected P815 cells were centrifuged, resuspended in 200 μl FCS, and labeled with 150 μCi of Na51CrO4 (NEN, Boston, MA) at 37°C, 5% CO2 for 1.5 h.

Skin grafting was conducted essentially as described (66). Briefly, recipients were anesthetized and shaved. Two graft beds were prepared with a skin bridge between them by removing two sections of skin from the posterior chest wall of each recipient. Full-thickness skin grafts (1–1.5 cm) harvested from the tails of donor and syngeneic control mice were engrafted onto the graft beds. Collodian was applied at the junction of the skin graft and donor skin to secure the grafts in place. Graft sites were covered with petroleum jelly gauze and a circumferential bandage, following which the mice were allowed to recover for 7 days. On day 7, the bandages were removed and the skin grafts were evaluated daily for redness, hair growth, hemorrhagic spots, presence of scales, and status of graft borders. Grafts were considered rejected when <10% of the graft bed contained viable grafted skin (66).

Statistical significance was determined with unpaired Student’s t test for the comparison of means with unequal variances (Microsoft Excel software; Redmond, WA). Differences between groups were considered to be significant if p < 0.05.

To further characterize and develop the HLA Tg model for studies of human MHC-dependent T cell recognition and responsiveness, we established a panel of TgM that expresses the HLA class I alleles HLA-A2, -B7, or -B27 in either the fully native form (i.e., HLAnat) together with hβ2m or as a hybrid molecule of the human peptide-binding α1/α2 domains linked to the mouse H-2Kb α3, transmembrane, and cytoplasmic domains (HLAhyb). Fig. 1 gives a schematic representation of the three HLAnat (Fig. 1,A) and three HLAhyb (Fig. 1 B) gene constructs. Of multiple founder mice generated and characterized, one representative line was selected for each construct for further detailed analysis and the studies reported here. Lines were selected on the basis of displaying an appropriate tissue distribution of expression at levels similar to each other and to endogenous H-2 class I.

Cell surface expression of Tg HLAnat and HLAhyb class I was analyzed for LNCs by flow cytometry with several anti-HLA class I mAbs, including B9.12.1 (reacts with all HLA class I alleles) (63), ME-1 (reacts with HLA-B7, -B27, and -Bw22) (61), and MA2.1 (reacts with HLA-A2 and -B17) (62). Endogenous H-2 class I expression was examined with the H-2Db-reactive mAb 28-14-8S (64). Following analysis of all Tg lines, single representative lines for each construct were selected for the studies described in this article on the basis of the most similar levels of Tg HLA expression to each other and to endogenous H-2 class I. As already described (5), surface expression of HLA-B7nat increased ∼10-fold in LNCs of mice coexpressing hβ2m (B7nat/hβ2 m), indicating suboptimal interaction of the human heavy chain with mouse β2m in mice Tg for the HLAnat class I gene alone. As a result, and to ensure the appropriate conformation of the Tg HLAnat heavy chains, the HLA-A2nat and HLA-B27nat TgM were derived by coinjection of fertilized eggs of the heavy chain gene along with the hβ2m gene. Fluorescent in situ hybridization analyses (67), as well as cotransmission of both the coinjected HLA and hβ2m genes to offspring, demonstrated that both genes were cointegrated at a single chromosomal site (results not shown). The HLA-B7nat/hβ2m mice were derived from the breeding of singly Tg parental HLA-B7nat and hβ2m lines (5). The HLAhyb gene constructs (Fig. 1 B) were designed such that the encoded molecules would associate much more efficiently than the HLAnat class I proteins with endogenous mouse β2m, thereby obviating the need for coexpression of hβ2m.

Fig. 2,A shows flow cytometry staining results for LNCs from HLA-A2nat/hβ2m and HLA-A2hyb TgM, whereas Fig. 2,B shows similar analyses for HLA-B7nat, -B7nat/hβ2m, -B7hyb, -B27nat/hβ2m, and -B27hyb TgM. Relative to the background levels of fluorescence observed for non-Tg LNCs stained with the same anti-HLA-A2.1 (Fig. 2,A), anti-HLA-B7/B27 (Fig. 2,B), or anti-pan-HLA class I (Fig. 2,C) mAbs, significant surface expression of both the native and hybrid molecules for each allele was detected on Tg cells (Fig. 2). A high level of hβ2m was also detected at the surface of cells from HLA-A2nat/hβ2m, -B7nat/hβ2m, and -B27nat/hβ2m TgM (not shown). The level of endogenous H-2Db (and Kb, not shown) class I expression was similar in all HLA Tg strains compared with non-TgM (Fig. 2 A, and not shown).

FIGURE 2.

Flow cytometric analysis of HLAnat and HLAhyb class I cell surface expression on Tg LNCs. A, LNCs from non-Tg (B6/SJL)F1 and HLA-A2nat/hβ2m and HLA-A2hyb class I TgM were stained with the HLA-A2-specific mAb MA2.1 (thick solid line) and the H-2Db-reactive mAb 28–14-8S (dashed dotted line). B, LNCs from non-Tg (B6/SJL)F1 and HLA-B7nat, HLA-B7nat/hβ2m, HLA-B7hyb, HLA-B27nat/hβ2m, and HLA-B27hyb class I TgM were stained with the HLA-B7/B27-specific mAb ME1 (thin solid line). C, LNCs from non-Tg (B6/SJL)F1 and HLAhyb (-A2, -B7, and -B27) TgM were stained with the pan-HLA class I-specific mAb B9.1.2.1 (dotted line). In all cases, the secondary Ab was FITC-conjugated F(ab′)2 goat anti-mouse IgG.

FIGURE 2.

Flow cytometric analysis of HLAnat and HLAhyb class I cell surface expression on Tg LNCs. A, LNCs from non-Tg (B6/SJL)F1 and HLA-A2nat/hβ2m and HLA-A2hyb class I TgM were stained with the HLA-A2-specific mAb MA2.1 (thick solid line) and the H-2Db-reactive mAb 28–14-8S (dashed dotted line). B, LNCs from non-Tg (B6/SJL)F1 and HLA-B7nat, HLA-B7nat/hβ2m, HLA-B7hyb, HLA-B27nat/hβ2m, and HLA-B27hyb class I TgM were stained with the HLA-B7/B27-specific mAb ME1 (thin solid line). C, LNCs from non-Tg (B6/SJL)F1 and HLAhyb (-A2, -B7, and -B27) TgM were stained with the pan-HLA class I-specific mAb B9.1.2.1 (dotted line). In all cases, the secondary Ab was FITC-conjugated F(ab′)2 goat anti-mouse IgG.

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A more quantitative estimate of the level of Tg HLA and H-2KbDb expression was previously determined for splenocytes for some of the mice shown in Fig. 2,B using Quantum Simply Cellular Microbeads (xxiv) (Sigma) to measure the number of binding sites with anti-HLA-B7/B27 mAb ME-1 and anti-H-2KbDb mAb HB-51 (39). These analyses showed that relative to non-Tg C57BL/6 (H-2b/b) and (B6/SJL)F1 (H-2b/s) mice, which carry ∼4 × 105 and 2 × 105 KbDb binding sites per cell, respectively, the levels of HLA-B7nat, -B7hyb, and -B27nat/hβ2m expression in the lines shown in Fig. 2,B were comparable, ranging from 1 to 3 × 105 binding sites per cell (39). As an alternate means of estimating the relative level of expression of each of the Tg HLAhyb alleles, LNCs from each line were stained with the monomorphic anti-HLA mAb B9.12.1. Fig. 2 C shows that the levels of HLA-A2hyb and -B7hyb were very similar to each other, whereas HLA-B27hyb was expressed at ∼30% of this level.

To evaluate the ability of non-Tg H-2-matched T cells to recognize and respond to Tg HLAnat and HLAhyb class I molecules as transplantation Ags in vitro, CML assays were performed. As the immune system of the responder strain used in these initial assays had not previously been exposed to the HLA molecule during immune development (i.e., in vivo), we consider this situation to represent a mouse anti-human MHC class I xenoresponse. Following a 5-day primary in vitro stimulation of non-Tg LNCs from (B6/SJL)F1 mice with spleen cells from each of the three HLAhyb or three HLAnat/hβ2m TgM, 51Cr release assays were performed using Con A-stimulated spleen cells from various non-TgM and TgM strains as targets. Fig. 3, A and D, shows the results obtained for the anti-HLAnat and anti-HLAhyb responses, respectively. For all alleles, the hybrid molecule (Fig. 3,D) induced a higher level of killing than its native counterpart (Fig. 3,A). This increase was most apparent for B7 and B27 because the native forms of these induced only very weak responses (Fig. 3,A). The elevated anti-HLAhyb response was not due simply to higher cell surface expression because flow cytometry (Fig. 2), quantitative measurements (39), and RNA analyses (not shown) showed that each of the HLAnat molecules in HLAnat/hβ2m TgM was expressed at similar or greater levels than the corresponding HLAhyb molecule (Fig. 2, A and B). In contrast to the very low level of killing detected for HLA-B7nat or -B27nat, the A2nat molecule induced a stronger response, which was further increased when A2 was expressed as a hybrid molecule (Fig. 3, A and D). The higher response to HLA-A2nat compared with -B7nat or -B27nat was not due to differences in expression level because B7nat/hβ2m was expressed at a higher level on the cell surface than A2nat/hβ2m, and B27nat/hβ2m expression was only slightly less (Fig. 2, A and B, and not shown). The magnitude of the anti-HLAhyb response for all alleles was close to that generated against the strong mouse class I allotransplantation Ag H-2Kbm1 (68, 69), whereas the response to HLA-A2nat was only slightly less (Fig. 3, A and D).

FIGURE 3.

Comparison of primary in vitro-stimulated CML response of non-Tg LNCs against Tg cells expressing xenogeneic HLAnat vs HLAhyb class I molecules. Responder LNCs from non-Tg (B6/SJL)F1 (H-2b/s) mice were stimulated in vitro with irradiated spleen cells from HLAnat class I (HLAnat/hβ2m) (A, B, and C) or HLAhyb class I TgM (HLAhyb) (D and E). The response against cells from mice carrying the H-2Kbm1 class I alloantigen was included for comparison in all experiments and is shown in (A). After 5 days of stimulation, the responding cultures were tested for specific cytotoxicity on target cells in 4-h 51Cr-release assays (65 ). A, (B6/SJL)F1 (H-2b/s) responders stimulated by HLA-A2nat, HLA-B7nat, or HLA-B27nat class I-expressing Tg cells (on a H-2b-matched background) were tested on Con A-stimulated mouse lymphoblast targets from HLA-A2nat, HLA-B7nat, or HLA-B27nat TgM (H-2b-matched), respectively. All anti-HLAnat responders were also tested on non-Tg targets and are shown for anti-HLA-A2nat responders. B, all three anti-HLAnat responding cultures were tested on HLA-A2nat-, HLA-B7nat-, or HLA-B27nat-expressing H-2-mismatched P815 (H-2d) cells respectively. Analogous experiments are shown in (D) and (E) for the anti-HLAhyb class I responders tested on HLAhyb-matched Con A-stimulated (D; H-2b-matched) or P815 (E; H-2d-mismatched) targets. C, responders stimulated by HLA-B7nat were tested on HLA-B7nat- and HLA-B7hyb-expressing P815 cells. The E:T ratio and the percentage of specific lysis are indicated on the x- and y-axes, respectively. The data represent the means of three individual experiments.

FIGURE 3.

Comparison of primary in vitro-stimulated CML response of non-Tg LNCs against Tg cells expressing xenogeneic HLAnat vs HLAhyb class I molecules. Responder LNCs from non-Tg (B6/SJL)F1 (H-2b/s) mice were stimulated in vitro with irradiated spleen cells from HLAnat class I (HLAnat/hβ2m) (A, B, and C) or HLAhyb class I TgM (HLAhyb) (D and E). The response against cells from mice carrying the H-2Kbm1 class I alloantigen was included for comparison in all experiments and is shown in (A). After 5 days of stimulation, the responding cultures were tested for specific cytotoxicity on target cells in 4-h 51Cr-release assays (65 ). A, (B6/SJL)F1 (H-2b/s) responders stimulated by HLA-A2nat, HLA-B7nat, or HLA-B27nat class I-expressing Tg cells (on a H-2b-matched background) were tested on Con A-stimulated mouse lymphoblast targets from HLA-A2nat, HLA-B7nat, or HLA-B27nat TgM (H-2b-matched), respectively. All anti-HLAnat responders were also tested on non-Tg targets and are shown for anti-HLA-A2nat responders. B, all three anti-HLAnat responding cultures were tested on HLA-A2nat-, HLA-B7nat-, or HLA-B27nat-expressing H-2-mismatched P815 (H-2d) cells respectively. Analogous experiments are shown in (D) and (E) for the anti-HLAhyb class I responders tested on HLAhyb-matched Con A-stimulated (D; H-2b-matched) or P815 (E; H-2d-mismatched) targets. C, responders stimulated by HLA-B7nat were tested on HLA-B7nat- and HLA-B7hyb-expressing P815 cells. The E:T ratio and the percentage of specific lysis are indicated on the x- and y-axes, respectively. The data represent the means of three individual experiments.

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As the above studies were performed using HLA Tg spleen-derived Con A-stimulated target cells sharing the same H-2b haplotype as the responding/effector cells (i.e., H-2b-matched), it was not possible to distinguish the extent to which the observed killing was due to recognition of intact HLA molecules directly as opposed to recognition of processed HLA-derived peptides presented by mouse H-2b MHC molecules. Therefore, this type of experiment was repeated using H-2-mismatched HLA class I gene-transfected mouse P815 (H-2d) cells as targets. Any killing observed for these targets must be due to direct recognition of intact HLA molecules. As shown in Fig. 3,E, non-Tg LNCs stimulated with HLA-A2hyb, -B7hyb, or -B27hyb spleen cells gave significant lysis of HLA-A2hyb-, -B7hyb-, or -B27hyb-expressing P815 cells, comparable to that observed for the Con A-stimulated targets (Fig. 3,D). Parental nontransfected P815 cells were not killed. Fig. 3,B shows that LNCs stimulated with Tg HLA-A2nat cells gave a modest but significant level of lysis of HLA-A2nat-P815 targets, whereas killing of HLA-B7nat-P815 cells by Tg B7nat-stimulated responders was only slightly above background and of HLA-B27nat-P815 targets by Tg B27nat-stimulated responders was undetectable. Therefore, the results of Fig. 3 demonstrate that, even when presented as xeno-Ags, all three HLAhyb alleles, and at least the HLA-A2nat molecule, are recognized largely, if not entirely, as intact MHC class I molecules by mouse T cells. This xeno-MHC recognition is also HLA allele-specific as the responding culture generated against one HLAhyb (or HLAnat) allele did not lyse targets expressing an alternate HLAhyb (or HLAnat) allele (not shown). Thus these xenoresponses are directed toward allele-specific polymorphisms as opposed to human-specific monomorphic determinants.

To investigate the basis of the apparent weak anti-HLAnat response further (Fig. 3, A and B), cultures derived from stimulating non-Tg (B6/SJL)F1 LNCs with HLA-B7nat Tg spleen cells were tested on HLA-B7hyb- vs HLA-B7nat-expressing P815 target cells (Fig. 3,C). As in Fig. 3,B, the killing of B7nat-P815 was very low and only slightly above the background level of lysis observed for parental P815 (not shown). In contrast, these same HLA-B7nat-stimulated cultures gave a much higher level of killing of HLA-B7hyb-P815 cells (Fig. 3 C). Similar experiments conducted with cultures derived from stimulating (B6/SJL)F1 LNCs with HLA-A2nat or -B27nat Tg spleen cells also showed a significantly increased level of killing of allele-matched HLAhyb-P815 compared with HLAnat-P815 targets (i.e., specific lysis increased from 34.6% for HLA-A2nat-P815 to 77.3% for A2hyb-P815 at an E:T ratio of 100:1 (p = 0.03), and from <10% for HLA-B27nat-P815 to 71.3% for B27hyb-P815 at an E:T ratio of 100:1 (p = 0.001)). This increased killing of allele-matched HLAhyb-expressing targets by HLAnat-stimulated (B6/SJL)F1 LNCs indicates that the low level of killing of HLAnat-expressing targets is not due to the absence of xenoreactive T cells in the normal non-Tg mouse repertoire able to recognize and be stimulated by the HLAnat molecule. Rather, the results suggest that some aspect of the structure of each HLAnat molecule on target cells important for mediating lysis is suboptimal compared with the HLAhyb molecule. This observation implies differential HLA domain-dependent interactions occurring at the induction vs effector phases of these in vitro CML assays.

To extend assessment of the relative immunogenicity of each of the HLAhyb and HLAnat class I alleles in vivo, tail skin from each Tg strain was grafted onto non-Tg H-2-matched ((B6/SJL)F1; H-2b/s) recipients, and the mean survival time (MST) was determined (Fig. 4). Although grafts from HLAhyb- or HLAnat-Tg-negative offspring (HLA Tgneg) or from mice Tg solely for hβ2m (hβ2m+) were indefinitely accepted (Fig. 4,A and Table I), grafts from HLA-A2hyb, -B7hyb, or -B27hyb TgM were rejected after 11.5 ± 2.4, 16.4 ± 1.8, and 17.2 ± 1.3 days, respectively (Fig. 4,B and Table I). These rates of rejection were similar to that for skin from mice carrying the known strong mouse class I alloantigen H-2Kbm1 (13.8 ± 1.5 days, Fig. 4,A), whereas a full MHC class I/class II strain mismatch (DBA/2; H-2d) was rejected slightly faster at about 9.4 ± 0.5 days (Fig. 4 A).

FIGURE 4.

Rejection of Tg HLAhyb and HLAnat class I tail-skin xenografts by H-2b-matched non-Tg recipients. Groups of non-Tg (B6/SJL)F1 recipient mice (see Table I for number of mice per group) were grafted with full-thickness skin from the following donor mice: HLAhyb or HLAnat transgene-negative [Tgneg] offspring from backcrosses of heterozygous HLA Tg parents with C57BL/6J, from hβ2m TgM (hβ2m+), from DBA/2 (H-2d), or from bm1 (H-2Kbm1) (A); HLA-A2hyb, -B7hyb, or -B27hyb TgM (B); HLA-A2nat/hβ2m, -B7nat, -B7nat/hβ2m, or -B27nat/hβ2m TgM (C). The plots show the percentage of mice within each group with surviving grafts as a function of days posttransplantation. The skin-graft protocol and criteria for rejection were as described in Materials and Methods.

FIGURE 4.

Rejection of Tg HLAhyb and HLAnat class I tail-skin xenografts by H-2b-matched non-Tg recipients. Groups of non-Tg (B6/SJL)F1 recipient mice (see Table I for number of mice per group) were grafted with full-thickness skin from the following donor mice: HLAhyb or HLAnat transgene-negative [Tgneg] offspring from backcrosses of heterozygous HLA Tg parents with C57BL/6J, from hβ2m TgM (hβ2m+), from DBA/2 (H-2d), or from bm1 (H-2Kbm1) (A); HLA-A2hyb, -B7hyb, or -B27hyb TgM (B); HLA-A2nat/hβ2m, -B7nat, -B7nat/hβ2m, or -B27nat/hβ2m TgM (C). The plots show the percentage of mice within each group with surviving grafts as a function of days posttransplantation. The skin-graft protocol and criteria for rejection were as described in Materials and Methods.

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Table I.

MST of xeno-HLA class I Tg skin grafts on non-Tg and various KO recipient mice

RecipientDonornaMST ± SD (days)b
Non-Tg (H-2bNon-Tg 42 >400 
 2>400 
 DBA/2 9.4 ± 0.5 
 H-2Kbm1 13.8 ± 1.5 
 A2nat/hβ211.5 ± 1.3 
 A2hyb 11.5 ± 2.4 
 B7nat 12.2 ± 1.8 
 B7nat/hβ211.6 ± 0.5 
 B7hyb 10 16.4 ± 1.8 
 B27nat/hβ210 10.5 ± 2.0 
 B27hyb 17.2 ± 1.3 
CD8−/− Non-tg 33 >300 
 H-2Kbm1 53, 57, >200× 5 
 A2nat/hβ29.5 ± 1.0 
 A2hyb 14.8 ± 3.7 
 B7nat/hβ211.5 ± 0.7 
 B7hyb 14.9 ± 2.1 
 B27nat/hβ211.8 ± 2.1 
 B27hyb 11 29.9 ± 8.1 
CD4−/− Non-tg 30 >300 
 A2nat/hβ214.2 ± 3.9 
 A2hyb 13.2 ± 1.3 
 B7nat/hβ211.3 ± 0.6 
 B7hyb 14.5 ± 2.9 
 B27nat/hβ213.8 ± 2.5 
 B27hyb 18.2 ± 4.4 
CD4−/−CD8−/− Non-tg 34 >300 
 A2nat/hβ210.5 ± 1.7 
 A2hyb 12.0 ± 1.3 
 B7nat/hβ227.2 ± 12.5 
 B7hyb 13.0 ± 1.7 
 B27nat/hβ222.0 ± 5.2 
 B27hyb 13.8 ± 1.3 
IgμM−/− Non-tg 34 >300 
 A2nat/hβ211.0 ± 0.0 
 A2hyb 10.3 ± 0.5 
 B7nat/hβ213.0 ± 0.0 
 B7hyb 11.6 ± 1.9 
 B27nat/hβ211.0 ± 0.7 
 B27hyb 12.7 ± 1.0 
RecipientDonornaMST ± SD (days)b
Non-Tg (H-2bNon-Tg 42 >400 
 2>400 
 DBA/2 9.4 ± 0.5 
 H-2Kbm1 13.8 ± 1.5 
 A2nat/hβ211.5 ± 1.3 
 A2hyb 11.5 ± 2.4 
 B7nat 12.2 ± 1.8 
 B7nat/hβ211.6 ± 0.5 
 B7hyb 10 16.4 ± 1.8 
 B27nat/hβ210 10.5 ± 2.0 
 B27hyb 17.2 ± 1.3 
CD8−/− Non-tg 33 >300 
 H-2Kbm1 53, 57, >200× 5 
 A2nat/hβ29.5 ± 1.0 
 A2hyb 14.8 ± 3.7 
 B7nat/hβ211.5 ± 0.7 
 B7hyb 14.9 ± 2.1 
 B27nat/hβ211.8 ± 2.1 
 B27hyb 11 29.9 ± 8.1 
CD4−/− Non-tg 30 >300 
 A2nat/hβ214.2 ± 3.9 
 A2hyb 13.2 ± 1.3 
 B7nat/hβ211.3 ± 0.6 
 B7hyb 14.5 ± 2.9 
 B27nat/hβ213.8 ± 2.5 
 B27hyb 18.2 ± 4.4 
CD4−/−CD8−/− Non-tg 34 >300 
 A2nat/hβ210.5 ± 1.7 
 A2hyb 12.0 ± 1.3 
 B7nat/hβ227.2 ± 12.5 
 B7hyb 13.0 ± 1.7 
 B27nat/hβ222.0 ± 5.2 
 B27hyb 13.8 ± 1.3 
IgμM−/− Non-tg 34 >300 
 A2nat/hβ211.0 ± 0.0 
 A2hyb 10.3 ± 0.5 
 B7nat/hβ213.0 ± 0.0 
 B7hyb 11.6 ± 1.9 
 B27nat/hβ211.0 ± 0.7 
 B27hyb 12.7 ± 1.0 
a

No. of recipient mice receiving the indicated skin graft.

b

Graft MST (in days) ± SD. In cases where all recipients failed to reject grafts from a certain donor strain, the MST is considered as greater than the number of days the mice were maintained (i.e., 200, 300, 400 days) until the experiment was terminated. In the case of CD8−/− KO recipients of bm 1 grafts, five of seven mice did not reject grafts by 200 days, whereas one rejected at 53 days and another at 57 days.

Based on the low level of response detected after in vitro stimulation with each of the HLAnat Tg class I molecules (Fig. 3, A and B), we expected skin grafts from these mice to be rejected slowly, if at all, by non-Tg recipients. However, the MST of grafts expressing HLA-B7nat with or without hβ2m on non-Tg recipients was found to be 11.6 ± 0.5 and 12.2 ± 1.8 days and for HLA-B27nat/hβ2m or HLA-A2nat/hβ2m grafts was 10.5 ± 2.0 and 11.5 ± 1.3 days (Fig. 4,C and Table I). For both B7 and B27, the rate of rejection of grafts expressing the HLAnat allele was significantly faster than grafts expressing the corresponding HLAhyb allele (p < 0.001). HLA-A2nat and -A2hyb-expressing grafts were rejected equally fast (MST of 11.5 days; see Fig. 4, B and C, and Table I).

In the case of the HLAhyb alleles, these results demonstrate that the immune system of non-TgM is able to recognize and respond to the xenogeneic human α1/α2 polymorphic domains of all three alleles such that graft rejection occurs at a similar rapid rate as for the strong allotransplantation Ag H-2Kbm1. In the case of the HLAnat alleles, despite only being recognized weakly in cytotoxicity assays in vitro, these molecules are equal or more potent than the corresponding HLAhyb alleles as xenotransplantation Ags in vivo.

Given the apparent discrepancy between the in vitro and in vivo results on recognition of Tg HLAnat vs HLAhyb class I molecules as transplantation Ags, we wished to examine the role of conventional cellular and humoral immune effector mechanisms in HLA Tg skin graft rejection. Therefore, HLAhyb or HLAnat class I Tg skin for each allele was grafted onto H-2b-matched gene-KO recipient mice that were deficient for either CD8+ T cells (CD8−/−KO) (52) (Fig. 5,A), CD4+ T cells (CD4−/−KO) (53) (Fig. 5,B), CD4+ and CD8+ T cells (CD8−/−CD4−/− double KO) (54) (Fig. 5,C), or B cells (IgμM−/−KO) (51) (Fig. 5,D). Although five of seven CD8−/−KO recipients retained mouse class I disparate H-2Kbm1 allografts for longer than 200 days (Fig. 5,A), grafts from TgM for all three HLAnat alleles as well as two of the three HLAhyb alleles (A2hyb and B7hyb) were rejected at rates (Fig. 5,A) very similar to those observed for the non-KO recipients (i.e., as in Fig. 4; see Table I). The only exception was that rejection of B27hyb skin was somewhat delayed to 29.9 ± 8.1 days compared with wild-type recipients (p = 0.01) (Fig. 5,A). Similarly, the allele-specific rejection rates of all HLAnat and HLAhyb grafts on CD4−/− and IgμM−/−KO recipients (Fig. 5, B and D; Table I) were virtually identical with those observed in the non-KO recipients. Grafts from TgM for all three HLAhyb alleles as well as HLA-A2nat/hβ2m were rejected by CD4−/−CD8−/− double KO recipients at rates similar to non-KO mice, whereas rejection of HLA-B7nat/hβ2m and -B27nat/hβ2m grafts were slightly delayed to 27.2 ± 12.5 days (p = 0.02) and 22.0 ± 5.2 days (p < 0.001) (Fig. 5,C and Table I). Taken together, these data suggest that although the relative contribution of CD8- and CD4-dependent T cell mechanisms to HLAhyb or HLAnat class I Tg skin graft rejection demonstrate some variability depending on the allele, neither CD8+ or CD4+ T cells or B cells are absolutely required for graft rejection. This lack of dependence on CD8+ T cells for rejection of HLAhyb grafts was particularly surprising in view of the in vitro CML results above as well as previous results of others (30, 36, 37).

FIGURE 5.

Rejection of Tg HLAhyb and HLAnat class I tail-skin xenografts by gene KO recipient mice. Groups of CD8−/− KO (A), CD4−/− KO (B), CD4−/− CD8−/− double KO (C), and IgμM−/− KO (D) mice (see Table I for number of mice per group) were grafted with tail skin from HLAnat (HLA-A2nat/hβ2m, -B7nat/hβ2m, -B27nat/hβ2m) or HLAhyb (HLA-A2hyb, -B7hyb, or -B27hyb) TgM, non-Tg siblings of the above HLA Tg donors (Non-Tg), or bm1 mice (H-2Kbm1). The plots show the percentage of mice within each group with surviving grafts as a function of days posttransplantation. The skin-graft protocol and criteria for rejection were as described in Materials and Methods.

FIGURE 5.

Rejection of Tg HLAhyb and HLAnat class I tail-skin xenografts by gene KO recipient mice. Groups of CD8−/− KO (A), CD4−/− KO (B), CD4−/− CD8−/− double KO (C), and IgμM−/− KO (D) mice (see Table I for number of mice per group) were grafted with tail skin from HLAnat (HLA-A2nat/hβ2m, -B7nat/hβ2m, -B27nat/hβ2m) or HLAhyb (HLA-A2hyb, -B7hyb, or -B27hyb) TgM, non-Tg siblings of the above HLA Tg donors (Non-Tg), or bm1 mice (H-2Kbm1). The plots show the percentage of mice within each group with surviving grafts as a function of days posttransplantation. The skin-graft protocol and criteria for rejection were as described in Materials and Methods.

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The above results show that rejection of HLAhyb-expressing grafts in vivo does not depend on CD8+ T cells. This contrasts to the clear CD8 dependence of rejection of grafts expressing a single mouse class I alloantigen (i.e., H-2Kbm1, Fig. 5 A). These observations may indicate that even when the “xeno” component of the foreign MHC molecule is limited to only the α1/α2 domains as in the HLAhyb molecules, there is still an inherent difference between the immune effector mechanisms that respond to this molecule compared with those that respond to an allo-MHC class I molecule. The HLA Tg model provides a unique opportunity to investigate this issue directly because it is possible to compare recognition of a given HLAhyb class I allele as a xenotransplantation Ag (i.e., by the immune system of non-TgM) with recognition of the identical molecule as an allotransplantation Ag (i.e., by the immune system of mice that are Tg for an alternate HLAhyb class I allele and thus developed in this human MHC-expressing environment). To this end, in vitro CML and in vivo graft rejection studies similar to the above were conducted, but rather than the responder LNCs and recipient mice being non-Tg, responder LNCs and graft recipients that expressed an alternate HLAhyb class I Tg allele relative to the stimulator and donor strain were used.

Following primary in vitro stimulation with HLA-B7hyb Tg spleen (Spl) cells, LNCs from HLA-matched B7hyb TgM did not lyse HLA-B7hyb-P815 targets above the background levels observed for P815 parental cells (Fig. 6,A). Compared with the strong xenogeneic anti-HLA-B7hyb response generated by non-Tg LNCs (Fig. 3, D and E), this lack of killing by B7hyb Tg LNCs indicates that CTL in these mice are tolerant to the HLA-B7hyb molecule. When LNCs from these same mice were stimulated with spleen cells from H-2Kbm1 mice and assayed on H-2Kbm1 targets, a high level of lysis was observed (Fig. 6,A) similar to that seen with non-Tg H-2b responders (Fig. 3,A). Following stimulation with HLA-B27hyb Tg spleen cells, LNCs from HLA-B7hyb TgM gave a significant level of lysis of both B27hyb Tg spleen Con A blast targets (B27hyb Tg Spl (H-2b)) and B27hyb-P815 targets (Fig. 6,B). This lysis appeared to be allele-specific as these same B27hyb-stimulated responders did not lyse HLA-A2hyb-expressing P815 cells (or A2hyb Tg spleen Con A-blasts; not shown) above background levels observed for parental P815 or C57BL/6J Con A blast targets (Non Tg Spl (H-2b); Fig. 6 B). These results demonstrate that the peripheral T cell repertoire of HLA-B7hyb TgM has become tolerant to the self-B7hyb allele but is alloreactive to alternate related human alleles such as HLA-B27hyb. Additional experiments of the peripheral repertoire of HLA-B27hyb TgM yielded similar results, demonstrating tolerance to the self-HLA-B27hyb allele and allele-specific alloreactivity to related non-self HLA alleles (i.e., B7hyb; not shown).

FIGURE 6.

Comparison of primary in vitro-stimulated CML response of HLAhyb/CD8+/+ and HLAhyb/CD8−/− KO LNCs against H-2-matched Tg cells expressing alternate HLAhyb class I alloantigens. Responder LNCs from HLA-B7hyb CD8+/+ mice (A and B) were stimulated in vitro with irradiated HLA-syngeneic (HLA-B7hyb) or non-Tg H-2Kbm1 (A) or HLA-allogeneic (HLA-B27hyb) (B) spleen cells and assayed on Con A-stimulated blasts or HLA-B7hyb-, HLA-B27hyb-, or HLA-A2hyb-expressing P815 target cells. Con A-stimulated blasts from C57BL/6J or bm1 mice or P815 cells were included as control targets. C, Responder LNCs from HLA-B7hyb/CD8−/− KO mice were stimulated with HLA-B27hyb spleen cells and assayed on HLAhyb-mismatched targets. D, Responder LNCs from non-Tg CD8−/− KO or HLA-B7hyb/CD8−/− KO mice were stimulated with irradiated H-2Kbm1 or HLA-B7hyb Tg spleen cells and assayed on bm1 Con-A blast targets or HLA-B7hyb-expressing P815 cells. The E:T ratios and the percentage of specific lysis are indicated on the x- and y-axes, respectively.

FIGURE 6.

Comparison of primary in vitro-stimulated CML response of HLAhyb/CD8+/+ and HLAhyb/CD8−/− KO LNCs against H-2-matched Tg cells expressing alternate HLAhyb class I alloantigens. Responder LNCs from HLA-B7hyb CD8+/+ mice (A and B) were stimulated in vitro with irradiated HLA-syngeneic (HLA-B7hyb) or non-Tg H-2Kbm1 (A) or HLA-allogeneic (HLA-B27hyb) (B) spleen cells and assayed on Con A-stimulated blasts or HLA-B7hyb-, HLA-B27hyb-, or HLA-A2hyb-expressing P815 target cells. Con A-stimulated blasts from C57BL/6J or bm1 mice or P815 cells were included as control targets. C, Responder LNCs from HLA-B7hyb/CD8−/− KO mice were stimulated with HLA-B27hyb spleen cells and assayed on HLAhyb-mismatched targets. D, Responder LNCs from non-Tg CD8−/− KO or HLA-B7hyb/CD8−/− KO mice were stimulated with irradiated H-2Kbm1 or HLA-B7hyb Tg spleen cells and assayed on bm1 Con-A blast targets or HLA-B7hyb-expressing P815 cells. The E:T ratios and the percentage of specific lysis are indicated on the x- and y-axes, respectively.

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Although CD8+ T cells are the main effectors responsible for rejection of H-2 class I-disparate skin allografts (Fig. 5,A), rejection of xeno-MHC class I grafts from HLAhyb or HLAnat TgM does not depend on either CD8+ or CD4+ T cells (Fig. 5, A and B). This was surprising given the high degree of amino acid and structural similarity of human and mouse MHC class I molecules (70, 71). It was unclear whether these results were due to an inherent difference in immune recognition mechanisms of xeno- vs allo-MHC in vivo even when the difference is limited solely to the α1/α2 domains as in the HLAhyb molecules, or rather reflects certain allele-specific and/or species-specific functional differences, or possibly other limitations of the HLA Tg model. To examine the role of CD8+ T cells in recognition of HLAhyb class I molecules as allo- vs xenotransplantation Ags, primary in vitro CML experiments were conducted as before but with responder LNCs from HLA-B7hyb TgM bred to homozygosity for the CD8α KO mutation and thus deficient for CD8+ T lymphocytes. Compared with the strong response of B7hyb/CD8+/+ LNCs (Fig. 6, upper panels) against both H-2Kbm1- (Fig. 6,A) and HLA-B27hyb- (Fig. 6,B) expressing stimulators and targets, LNCs from HLA-B7hyb/CD8−/−KO mice (Fig. 6, lower panels) were unable to mount cytotoxic responses to either of these mouse (bm1) (Fig. 6,D) or human (B27hyb) (Fig. 6,C) alloantigens above background. CD8−/− KO mice that did not carry the HLA transgene were also unable to respond to H-2Kbm1 (Fig. 6 D). Additional studies using LNCs from HLA-B27hyb/CD8−/−KO (instead of HLA-B7hyb/CD8−/−KO) mice as a source of responders gave analogous results (not shown). Therefore, similar to the in vitro response to mouse class I alloantigens such as H-2Kbm1, CD8+ T cells that develop in HLAhyb TgM are able to recognize and respond to alternate human MHC HLAhyb class I alleles as alloantigens.

Although CD8+ T cells from non-Tg (xeno) and HLAhyb Tg (allo) mice are able to respond in vitro to Tg cells expressing a foreign HLAhyb allele, CD8+ cells are not required for the rapid xenorejection by non-TgM of grafts from HLAhyb TgM in vivo. To examine whether expression of the HLA class I molecule as a self-MHC allele influences the mechanisms by which the immune system responds in vivo to grafts from mice expressing alternate HLA class I alleles (i.e., human allo-MHC Ags), a series of skin-grafting experiments were conducted (Fig. 7). Compared with rejection of (xeno) HLA-B27hyb skin grafts by non-TgM at about 17.2 ± 1.3 days (Fig. 4,B, Table I), HLA-B27hyb TgM accepted HLA-B27hyb (i.e., syngeneic) Tg skin indefinitely (Fig. 7,A, Table II). However, these same animals rejected grafts from both HLA-B7hyb and -A2hyb TgM at rates similar to those for non-Tg recipients (i.e., 17.2 ± 4.0 and 11.7 ± 2.9 days for HLA-B27hyb recipients (Fig. 7,A, Table II) vs 16.4 ± 1.8 and 11.5 ± 2.4 days for non-Tg recipients (Table I)). A similar pattern was seen for HLA-B7hyb TgM in that, compared with non-TgM, which rejected B7hyb grafts at 16.4 ± 1.8 days (Table I), B7hyb recipients indefinitely accepted these grafts (Table II) but rejected HLA-B27hyb and -A2hyb Tg skin after 17.3 ± 3.4 and 13.3 ± 2.9 days (Fig. 7,B, Table II) (compared with 17.2 ± 1.3 and 11.5 ± 2.4 days, respectively, for non-Tg recipients).

FIGURE 7.

Rejection of Tg HLAhyb allele- or locus-mismatched class I tail-skin allografts by HLAhyb/CD8+/+ and HLAhyb/CD8−/− recipients. A and B, Results of grafting HLA-B27hyb/CD8+/+ (A) or HLA-B7hyb/CD8++ (B) mice with HLAhyb allele-matched or HLAhyb allele- or locus-mismatched skin grafts. C and D, Results of grafting HLA-B27hyb/CD8−/− (C) or HLA-B7hyb/CD8−/− (D) mice with HLAhyb-allele-mismatched (B7hyb, C; B27hyb, D) or HLAhyb-locus-mismatched (A2hyb, C and D) Tg skin grafts. The plots show the percentage of mice within each group with surviving grafts as a function of time (days) posttransplantation. The skin-graft protocol and criteria for rejection were as described in Materials and Methods. See Table II for number of recipient mice per group and graft MSTs.

FIGURE 7.

Rejection of Tg HLAhyb allele- or locus-mismatched class I tail-skin allografts by HLAhyb/CD8+/+ and HLAhyb/CD8−/− recipients. A and B, Results of grafting HLA-B27hyb/CD8+/+ (A) or HLA-B7hyb/CD8++ (B) mice with HLAhyb allele-matched or HLAhyb allele- or locus-mismatched skin grafts. C and D, Results of grafting HLA-B27hyb/CD8−/− (C) or HLA-B7hyb/CD8−/− (D) mice with HLAhyb-allele-mismatched (B7hyb, C; B27hyb, D) or HLAhyb-locus-mismatched (A2hyb, C and D) Tg skin grafts. The plots show the percentage of mice within each group with surviving grafts as a function of time (days) posttransplantation. The skin-graft protocol and criteria for rejection were as described in Materials and Methods. See Table II for number of recipient mice per group and graft MSTs.

Close modal
Table II.

MST of HLA class I Tg skin grafts on HLA-CD8+/+ and HLA-CD8−/− recipient mice

RecipientDonornaMST ± SD (days)b
B27/Kb-CD8+/+ B27hyb >200 
 A2hyb 11.7 ± 2.9 
 B7hyb 17.2 ± 4.0 
B7/Kb-CD8+/+ B7hyb >200 
 A2hyb 13.3 ± 2.9 
 B27hyb 17.3 ± 3.4 
    
B27/Kb-CD8−/− B27hyb >200 
 A2hyb 12.4 ± 1.7 
 B7hyb >200 
B7/Kb-CD8−/− B7hyb >200 
 A2hyb 20.8 ± 3.9 
 B27hyb >200 
RecipientDonornaMST ± SD (days)b
B27/Kb-CD8+/+ B27hyb >200 
 A2hyb 11.7 ± 2.9 
 B7hyb 17.2 ± 4.0 
B7/Kb-CD8+/+ B7hyb >200 
 A2hyb 13.3 ± 2.9 
 B27hyb 17.3 ± 3.4 
    
B27/Kb-CD8−/− B27hyb >200 
 A2hyb 12.4 ± 1.7 
 B7hyb >200 
B7/Kb-CD8−/− B7hyb >200 
 A2hyb 20.8 ± 3.9 
 B27hyb >200 
a

No. of recipient mice receiving the indicated skin graft.

b

Graft MST (in days) ± SD. In cases where all recipients failed to reject grafts from a certain donor strain, the MST is considered as greater than the number of days the mice were maintained (i.e., 200, 300, 400 days) until the experiment was terminated.

The above results demonstrate that HLA-B7hyb and HLA-B27hyb class I TgM become tolerant to the α1 and α2 domains of the self-Tg HLA allele but are reactive to the corresponding domains in alternate class I HLA-B alleles (are alloreactive) as well as in alternate locus products (i.e., HLA-A2hyb). To evaluate the requirement for CD8+ T cells in rejection of these HLAhyb-expressing Tg allografts, HLA-B27hyb and -B7hyb mice that were bred to be deficient for CD8 expression were transplanted with HLA-B27hyb, -B7hyb, or -A2hyb Tg skin. HLA-B27hyb/CD8−/− recipients did not reject allele-matched (B27hyb) or allele-mismatched (B7hyb) grafts but did reject locus-mismatched (A2hyb) grafts at a rate similar to that for B27hyb/CD8+/+ TgM and non-TgM (i.e., 12.4 ± 1.7 vs 11.7 ± 2.9 vs 11.5 ± 2.4 days). Similarly, HLA-B7hyb/CD8−/− recipients did not reject skin expressing either self (i.e., B7hyb) or non-self (B27hyb) HLA-B Tg alleles but did reject skin from HLA-A2hyb TgM (Fig. 7, C and D; Table II). These results demonstrate that rejection of Tg skin expressing alternate HLA class I B alleles depends exclusively on CD8+ T cells, whereas rejection of locus-mismatched HLA Tg skin depends on other CD8 T cell-independent mechanisms. Rejection of Tg skin expressing any of the three HLA alleles by non-TgM (i.e., as xenotransplantation Ags) was also found to occur through a CD8 T cell-independent route.

Although HLA TgM have the potential to provide a useful model for studies of human MHC-dependent immune function in vivo, the extent to which mouse T cells are able to functionally respond to human class I or II alleles in this system was not clear (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48). We established a panel of TgM carrying different HLA alleles in both the fully human (HLAnat) and hybrid human/mouse (HLAhyb) forms to address several aspects of immune recognition to clarify this issue. Here we have examined whether the same in vitro and in vivo immune mechanisms are responsible for recognition of a given MHC class I allele as a xeno- as opposed to an allotransplantation Ag. To minimize any influence of quantitative differences in expression level on differential function of the three HLA alleles in either the native or hybrid form, we selected single lines for each that expressed the most similar cell surface levels of each Tg product to each other and to endogenous mouse H-2 class I (Fig. 2). The close to physiological expression levels of all Tg HLAhyb and HLAnat molecules, together with reactivity with all anti-HLA class I mAbs specific for polymorphic or monomorphic determinants tested, implies that each Tg HLAhyb and HLAnat (in the presence of hβ2m) molecule is able to efficiently undergo intracellular trafficking and association with endogenous peptides and to adopt an appropriate conformation at the cell surface.

Despite physiological surface levels of Tg HLAnat class I, the in vitro cytotoxicity studies in Fig. 3, A and B, together with previous results of our own and others, implied a limitation at some level in the efficiency of recognition of fully human MHC class I molecules by non-Tg cytotoxic T cells. To investigate this issue, a number of groups, including ours, have studied TgM expressing HLAhyb Tg constructs to distinguish whether inefficient xenorecognition of HLAnat class I was due to the low frequency of mouse TCRs able to interact with the HLA class I α1/α2 domains (i.e., “holes” in the repertoire) as opposed to other species-specific molecular incompatibilities involving interactions outside the peptide-binding α1/α2 domains. The results in Fig. 3, D and E, demonstrate that the in vitro primary xenoresponse of non-TgM T cells to HLA class I molecules is enhanced for all three alleles when cells from HLAhyb rather than HLAnat TgM are used as stimulators and targets. In the HLAhyb form, all alleles induced levels of killing that were close to that against the strong mouse class I transplantation Ag H-2Kbm1 (68, 69). In the HLAnat form, only HLA-A2 induced a reproducibly significant level of killing. It is unclear why this HLAnat allele induces a stronger response than the others but, given the similar cell surface expression levels, it must be due to structural polymorphic differences. This type of HLAnat allelic difference may explain some of the differing results reported by groups working with various HLAnat class I Tg strains.

With regard to the non-Tg response against HLAhyb class I, it is important to recognize that the strong lysis of H-2-mismatched HLAhyb-transfected P815 targets indicates that killing is due largely to direct recognition of intact MHC molecules. The induced responses were also allele-specific, indicating that they were directed primarily at the polymorphic regions as opposed to shared human-specific determinants. Together, these results imply that the weak anti-HLAnat response detected in vitro is not due to a low frequency of xeno-MHC reactive mouse T cells in the non-Tg repertoire, but rather mainly to species-specific interactions outside of the Ag-binding cleft. A similar conclusion was reached previously by others studying HLA-A2 (30, 37) and -B27 (36), but not for -B7 (41).

Although HLAnat-stimulated non-Tg responders were unable to give significant levels of killing of HLAnat allele-matched targets, these same responders gave much higher levels of killing of HLAhyb allele-matched targets for HLA-B7 (Fig. 3,C), -A2, and -B27 (not shown). These results suggest that the low killing observed in the experiments of Fig. 3, A and B, is not necessarily because the xeno-HLAnat molecules are unable to induce a cellular response but rather may be due in part to a suboptimal ability of the induced cells to recognize the HLAnat molecule on target cells. In studies of the anti-influenza T cell response in analogous strains of HLA-A2nat and A2/Kb TgM, Sherman et al. (37) observed that A2nat-restricted effectors from HLA-A2nat TgM could lyse A2Kb (A2hyb) targets in the presence or absence of flu peptide. The interpretation of this result was that the A2nat-restricted CTLs had only low affinity for the A2nat molecule but an increased affinity for the A2Kb (A2hyb) molecule as a result of incorporating mouse CD8 in the interaction by inclusion of the mouse α3 domain in the A2hyb molecule. Although our studies have examined the non-Tg T cell response to xeno-HLA-A2 rather than the usage of HLA-A2 as a restriction element by Tg T cells, the killing of HLA-A2hyb targets by HLA-A2nat-induced T cells may also be the result of an increased affinity of interaction resulting from involvement of mouse CD8 with the A2hyb vs A2nat molecule during the killing of HLA-A2hyb vs A2nat targets.

It is generally accepted that in vitro CML assays used above are an in vitro correlate of MHC class I disparate graft rejection in vivo (72, 73, 74, 75). Based on this, it would be expected that if recognition of HLA Tg class I molecules as xenotransplantation Ags by the non-TgM immune system is similar to that of mouse H-2 class I molecules, then the mechanisms mediating HLA Tg and H-2 class I disparate graft rejection should be similar and be reflected by these assays. However, based on our skin graft rejection studies, we believe that some of the previous inconsistencies among studies using this type of model result from the apparent breakdown of this correlation. For example, given the very low level of killing observed following stimulation with each Tg HLAnat product (Fig. 3), the rapid rejection of skin grafts from these same HLAnat TgM was not expected (Fig. 4 C). However, although Van Twuyver et al. (76) also reported rapid rejection of skin grafts from mice Tg for HLA-B27nat, others appear to have limited their analyses to in vitro primary CML assays with the assumption that the in vitro results reflected in vivo graft recognition and rejection (36).

The mechanisms underlying the very rapid rejection of HLAnat Tg grafts are not obvious. Clearly, it is not due to the influence that coexpressing hβ2m has on either the quantitative level of expression or the conformation of the HLAnat class I heavy chain as skin grafts from singly Tg HLA-B7nat mice (i.e., hβ2m-negative) are also rapidly rejected (Fig. 4,C) and those from singly Tg hβ2m mice (i.e., HLA-negative/hβ2m-positive) are indefinitely accepted (Fig. 4 A). Also, the use of gene KO graft recipient mice deficient for either CD8+ or CD4+ T cells or B cells showed that these populations on their own were not responsible for rejection of Tg HLAnat grafts. Either other effector mechanisms (i.e., NK cells), or multiple mechanisms as suggested by the somewhat prolonged survival of grafts from two of the three HLAnat Tg strains in CD4−/−CD8−/− double KO recipients, are operative in the rapid rejection of these grafts. Studies to investigate these possibilities are in progress.

The second instance in which there is a discrepancy between results obtained from in vitro CML vs in vivo graft rejection assays was with xenorecognition of the HLAhyb class I molecules. The in vitro CML assays shown here, together with results from others (36), demonstrate that each HLAhyb class I allele is recognized as efficiently as the mouse class I alloantigen bm1, presumably due to improved interaction of the HLAhyb, compared with the HLAnat class I molecule, with mouse CD8 as a result of including the mouse α3 domain (36, 37). However, our results showed that when CD8−/− KO mice were engrafted with donor HLAhyb Tg skin, the allele-specific rejection rates (Fig. 5,A) were very similar to those observed for wild-type (CD8+/+) recipients. Thus, despite the differences detected in vitro for the HLAnat and HLAhyb xeno-MHC molecules, these effects are of no significance in vivo in at least two respects: first, despite poor recognition in vitro, HLAnat grafts are rejected very rapidly in vivo at rates that are equal to or faster than those for the corresponding HLAhyb grafts or even bm1 grafts; and second, despite improved recognition in vitro of HLAhyb class I molecules by non-TgM CD8+ T cells, this effect is completely irrelevant to rejection of HLAhyb grafts in vivo. In contrast, the absence of CD8+ T cells had a significant effect on the survival of H-2Kbm1 skin grafts. This finding of prolonged survival of mouse class I allogeneic, but not HLAhyb class I Tg, skin grafts in CD8−/− KO recipients argues that CD8+ T cells play an important role in the rejection of murine allografts disparate at a single MHC class I molecule but not in class I-disparate xenografts. A similar independence of HLAhyb Tg graft rejection on CD4+ T cells was revealed when CD4−/− KO recipients were grafted with HLA Tg skin (Fig. 5 B). Thus, similar to HLAnat grafts, these data indicate that neither CD8+ or CD4+ T cells on their own are necessary for rejection of xeno-HLAhyb Tg grafts and that either other or multiple mechanisms must be involved. As the only difference between the HLAhyb molecules and the “self” H-2Kb class I molecule is in the α1/α2 domains, our results show that although the human (xeno) α1/α2 domains are sufficient for recognition as a very strong major histocompatibility transplantation Ag, the immune mechanisms induced by this xeno-MHC class I molecule are distinct from those induced by an allo-MHC (mouse) class I molecule.

The strong non-Tg anti-HLAhyb xenoresponses detected in vitro and in vivo were not detected when immune cells from HLAhyb TgM were stimulated with HLAhyb allele-matched cells (Fig. 6,A) or grafts (Fig. 7, A and B). In contrast, stimulation of HLAhyb Tg cells with HLAhyb allele-mismatched (i.e., allogeneic) Tg cells led to strong lysis of target cells expressing the mismatched HLAhyb allele but not the HLAhyb self allele or an alternate third party allele (Fig. 6, A and B). Similarly, although HLAhyb TgM were tolerant to grafts expressing their self-HLAhyb allele, they rapidly rejected grafts from mice expressing a HLAhyb allele-mismatched (rejection of B7hyb grafts by B27hyb TgM, Fig. 7,A; rejection of B27hyb grafts by B7hyb TgM, Fig. 7,B) as well as locus-mismatched grafts (rejection of HLA-A2hyb grafts by -B27hyb TgM, Fig. 7,A; and by -B7hyb TgM, Fig. 7 B). Thus, given that the non-Tg response to HLAhyb class I molecules as xeno-MHC Ags involves both a CD8+ T cell-dependent component (based on the in vitro CML assays) and a non-CD8+/non-CD4+ T cell/non-B cell-dependent component (based on the graft-rejection studies), allele-specific tolerance in each of these compartments develops to the xenogeneic HLA class I α1/α2 domains as a result of expressing the HLAhyb allele as a Tg product during immune development.

In contrast to the xeno-MHC skin graft experiments in which rejection occurred despite the absence of conventional T or B cells (Fig. 5), both in vitro recognition and in vivo rejection of HLA-Bhyb allele-mismatched cells or skin by HLA-Bhyb TgM (i.e., B7hyb anti-B27hyb or B27hyb anti-B7hyb) was absolutely dependent on the presence of CD8+ T cells (Fig. 6 and 7). This CD8 dependence appears to be analogous to that seen for the response of H-2b mice to the murine alloantigen H-2Kbm1 and suggests that the HLA-B7hyb and -B27hyb molecules are recognized as true alloantigens by the immune systems of HLA-B27hyb and -B7hyb TgM, respectively. Interestingly, rejection of HLAhyb locus-mismatched (i.e., HLA-A2hyb) skin by either HLA-B27hyb (Fig. 7,C) or -B7hyb (Fig. 7 D) TgM does not depend on CD8+ T cells, similar to xenorejection of skin from any of the HLAhyb Tg strains by non-TgM.

Taken together, our results allow the following conclusions to be made: 1) recognition of single HLA class I molecules in vivo as xenotransplantation Ags depends on multiple immune effector mechanisms that do not include CD8+ T cells; 2) expression of these xeno-HLAhyb class I molecules in TgM as self-MHC leads to tolerance of all of these effector mechanisms; 3) recognition of HLAhyb locus-matched alternate alleles (i.e., B7 vs B27) by HLAhyb TgM depends exclusively on CD8+ T cells in a manner analogous to endogenous mouse H-2 class I alloantigens; and 4) recognition of HLAhyb locus-mismatched alternate alleles (i.e., HLA-B vs HLA-A) by HLAhyb TgM does not depend on CD8+ T cells and implies that, despite being human, due to their reduced homology to the self-HLA allele, may be recognized essentially as a xeno-MHC class I. It seems likely that similar immune recognition events take place in the human in response to HLA-mismatched grafts but, because of the increased complexity, they have been difficult to discern. As such, the results presented here should have relevance for development of protocols for modulating graft rejection in human organ transplantation.

1

This work was supported, in part, by the Canadian Universities Research Fund, the National Cancer Institute of Canada, and the Research Institute of The Hospital For Sick Children. S.H.B. is a recipient of support from The Hospital for Sick Children Restracom. J.W.C. is a scholar of the Medical Research Council of Canada.

3

Abbreviations used in this paper: β2m, β2-microglobulin; hβ2m, human β2m; HLA-A2/Kb or -A2hyb, hybrid HLA-A2/H-2Kb class I molecule; HLA-B7/Kb or -B7hyb, hybrid HLA-B7/H-2Kb class I molecule; HLA-B27/Kb or -B27hyb, hybrid HLA-B27/H-2Kb class I molecule; LNCs, lymph node cells; MST, mean survival time; A2nat, native (entirely human) HLA-A2 class I molecule; B7nat, native (entirely human) HLA-B7 class I molecule; B27nat, native (entirely human) HLA-B27 class I molecule; Tg, transgenic; TgM, Tg mouse/mice; KO, knockout; CML, cell-mediated lympholysis.

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