Joyce Swensson

Characteristics of immunoglobulin gene usage of the xenoantibody binding to gal-alpha(1,3)gal target antigens in the gal knockout mouse.

BACKGROUND Natural antibodies that react with galactose-alpha(1,3)galactose [galalpha(1,3)gal] carbohydrate epitopes exist in humans and Old World primates because of the inactivation of the alpha1,3-galactosyltransferase (alpha1,3GT) gene in these species and the subsequent production of antibodies to environmental microbes that express the galalpha(1,3)gal antigen. The Gal knockout (Gal o/o) mouse, produced by homologous disruption of the alpha1,3GT gene, spontaneously makes anti-galalpha(1,3)gal antibodies and can be used to study the genetic control of humoral immune responses to this carbohydrate epitope. METHODS Six hybridomas that produce monoclonal antibodies (mAbs) to galalpha(1,3)gal were generated in Gal o/o mice. The mAbs were tested to characterize the binding activity with flow cytometry using pig aortic endothelial cells and ELISA with galalpha(1,3)gal carbohydrates. The VH and VK genes of these hybridomas were cloned, sequenced, and analyzed. RESULTS The mAbs showed distinct patterns of antibody binding to galalpha(1,3)gal antigens. The VH genes that encode the mAb binding activity were restricted to a small number of genes expressed in their germline configuration. Four of six clones used closely related progeny of the same VH germline gene (VH441). Comparison of the mouse gene VH441 to the human gene IGHV3-11, a gene that encodes antibody activity to galalpha(1,3)gal in humans, demonstrates that these two genes share a nonrandom distribution of amino acids used at canonical binding sites within the variable regions (complimentary determining regions 1 and 2) of their immunoglobulin VH genes. CONCLUSIONS These results demonstrate the similarity of the Gal o/o mice and humans in their immune response to galalpha(1,3)gal epitopes. Gal o/o mouse can serve as a useful model for examining the genetic control of antibody/antigen interactions associated with the humoral response to pig xenografts in humans.

Genetic control of the humoral responses to xenografts. III. Identification of the immunoglobulin V(H) genes responsible for encoding rat immunoglobin G xenoantibodies to hamster heart grafts.

BACKGROUND We have previously reported that the early phases of the immune response of rats to hamster xenografts are characterized by the production of IgM xenoantibodies encoded by a restricted group of Ig germline V(H) genes (V(H)HAR family). In the later phases of the reaction, an IgM to IgG isotype switch occurs and our study examines the structure of the rearranged V(H)HAR genes used to encode IgG antibodies after this isotype switch. METHODS A quantitative polymerase chain reaction was used to investigate the changes in the levels of V(H)HAR+ IgG mRNA seen after xenotransplantation. cDNA libraries specific for V(H)HAR+ Iggamma chain were established from total RNA extracted from splenocytes of naive rats and xenograft recipients of hamster hearts at days 4, 8, 21, and 28 posttransplantation. Colony filter hybridization was used to estimate the relative frequency of the use of individual V(H)HAR+ IgG subclasses. Selected IgG clones from day 21 cDNA libraries were sequenced and analyzed for VH-D-J(H) gene usage and antibody combining site structure. RESULTS The level of mRNA for V(H)HAR+ IgG increased 6-fold in xenograft recipients at day 21 post-transplantation when compared with naive animals. The relative frequency of isotype usage for V(H)HAR+ IgG1 antibodies alone increased from 22.3% at day 0 to 37.4% at day 21 PTx. Ten IgG clones from the day 21 cDNA libraries have been sequenced for the rearranged V(H)-D-J(H) genes. Thirty percent (3/10) of these IgG clones used V(H)HAR genes for the coding of heavy chain variable region with limited numbers of nucleic acid substitutions (>98% identity with their germline progenitors) although others demonstrated increased variation in nucleotide sequences (95-97% identity) when compared with germline V(H) genes. Analysis of the canonical binding site structure from the predicted amino acid sequences demonstrated that the majority of IgG clones (9/10) displayed a similar pattern of conserved configurations for their combining sites. CONCLUSIONS The change in IgM to IgG antibody production in the later stages of the humoral immune response of rats to hamster xenografts is associated with an IgM to IgG isotype switch and an increased production of antibodies of the IgG1 isotype. Rat anti-hamster IgG xenoantibodies continue to express the V(H)HAR family of V(H) genes, many in their original germline configuration, to encode antibody recognition of the hamster target antigens. There are, however, a majority of antibodies for which the V(H) genes express evidence of increased nucleic acid sequence variation when compared to currently available germline sequences. The source of this variation is not known but may represent the expression of as yet unidentified germline genes and/or the introduction of T cell-driven somatic mutations. Despite the appearance of this variation, the unusual level of conservation in key antigen binding sites within the V(H) region suggests the variation, independent of its origin, may have a limited influence on the restricted nature of the host antibody response to xenografts.

Human serum reactivity to porcine endothelial cells after antisense-mediated down-regulation of GpIIIa expression

The hyperacute rejection of vascularized grafts exchanged between discordant species is a result of the binding of preformed natural antibodies to the endothelium of the donor organ, and the subsequent activation of the complement system. Human natural antibodies to pig endothelial cell antigens appear to be predominantly directed at carbohydrate epitopes expressed by a variety of porcine integrins, including GpIIIa. The identification of porcine xenoantigens whose recognition by human natural antibodies results in hyperacute rejection would allow for the development of strategies to genetically modify the xenograft reaction. We have used antisense technology to down-regulate the expression of one of seven recently identified xenoantigens from the surface of pig aortic endothelial cells. Down-regulation of GpIIIa on endothelial cells resulted in a 20.8% decrease in the mean channel shift (MCS) of IgM natural antibody binding from pooled human sera, and a 28-35% decrease in the MCS of IgM binding from two high-titer individuals. The MCS for human IgG natural antibody binding to the surface of pig cells decreased by 27%. Natural antibody-mediated cytotoxicity to pig endothelial cells was not significantly altered, as indicated by a 2.5-6% decline in complement-mediated cytotoxicity. These results indicate that down-regulation of GpIIIa alone may not be sufficient to significantly alter xenograft rejection. Our results also suggest, however, that antisense-mediated regulation of a functionally important target antigen is technically feasible and may represent a strategy to prevent the xenograft reaction.

Failure of anti-Forssman antibodies to induce rejection of mouse heart xenografts.

The Forssman antigen has been proposed to be a target for the xenograft reaction in selected species combinations, including the rat and mouse, which are Forssman‐negative and ‐positive species respectively. The mouse represents an important experimental model for a variety of immune‐mediated disease processes, and the availability of a simple, inexpensive target antigen could provide an important tool for studying a selected portion of the immunologic basis for the rejection of xenografts. We have examined the potential that antibodies directed against mouse Forssman antigen could cause the hyperacute rejection of mouse heart xenografts in naive rat recipients. The Forssman antibodies tested included rat anti‐mouse (R‐anti‐M) antiserum, R‐anti‐M antiserum depleted of anti‐Forssman (anti‐F) antibodies, rat anti‐sheep red blood cell (SRBC) antiserum containing anti‐F antibodies and a rat monoclonal anti‐F IgM antibody. Our results demonstrate that the R‐anti‐M antiserum at day 4 post transplantation displayed significant titers (1:512–4096) of hemagglutinating antibodies for SRBC and mild to moderate levels of IgM that specifically binds to Forssman glycolipid (GalNAcαl–3GalNAcβl–3Galαl–4Galβ1–4Glcβ1–1ceramide) as measured by an enzyme‐linked immunosorbent assay (ELISA). Passive transfer of the R‐anti‐M serum to rats receiving mouse cardiac grafts immediately after transplantation caused hyperacute rejection of the xenografts. Sequential immunoabsorption of R‐anti‐M sera with SRBCs resulted in total removal of the anti‐Forssman activity (as defined by negative hemagglutination titer and minimal binding to Forssman glycolipid in ELISA). The anti‐F Ab‐depleted R‐anti‐M antisera, however, retained the capacity to induce hyperacute rejection of the mouse hearts [n = 6, median survival time (MST) 13 min] when passively transferred to rat recipients. Anti‐Forssman antibodies induced by immunization of LEW rats with SRBCs or a rat anti‐Forssman monoclonal antibody, mAb M.1.22.25, exhibited substantial anti‐Forssman activity (hemagglutinating titer 1: 512–4096 and moderate‐to‐strong binding to Forssman glycolipid in ELISA respectively). These antibodies also failed, however, to trigger hyperacute rejection of mouse cardiac xenografts. In conclusion, our results suggest that the rat anti‐Forssman antibodies, including those stimulated by mouse cardiac xenografts, do not appear to play a role in the immediate (hyperacute) rejection of mouse heart xenografts.

Maturation of xenoantibody gene expression during the humoral immune response of rats to hamster xenografts.

Abstract: Immunoglobulin isotype switching represents an important component of antibody maturation in the development of humoral immune responses. We have recently conducted a series of studies in a nonimmunosuppressed rodent model to define the kinetics of xenoantibody production and seek evidence for the maturation of xenoantibody Ig gene expression by xenograft recipients. LEW rats were transplanted with hamster cardiac xenografts and the grafts were allowed to remain in situ for prolonged immune stimulation of the host. Anti‐hamster antibodies were examined at days 4, 8, 21, 28 and 40 post‐transplantation. cDNA libraries specific for rat µ or γ heavy chains were constructed from B lymphocytes of the xenograft recipients at day 4 and day 21 post‐transplantation. Selected cDNA clones encoding the Ig VHHAR family of genes from each group were sequenced and analyzed for the presence of somatic mutations. We found that the reactivity of xenoantibodies examined with flow cytometry underwent sequential changes in which IgM titers peaked at day 8 post‐transplantation (PTx) and returned to low levels after 21 days. IgG titers started to increase at about one week PTx and peaked at 21–28 days. All the IgG isotypes (IgG1, 2a, 2b and 2c) were differentially involved in the IgG responses. Serum passive transfer experiments demonstrated that IgM antibody fractions separated from sera at day 4 post‐transplantation were capable of causing hyperacute rejection (HAR) of hamster xenografts, whereas IgM fractions from days 21–40 failed to cause HAR (N = 7, MST = 4 days), a pattern that was consistent with a rise in total xenoreactive IgM levels at days 4–8 and a fall to low levels at 21 days post‐transplantation. IgG‐containing fractions separated from day 21–40 antisera caused HAR (N = 7, MST = 36 min) whereas IgG fractions from day 8 sera failed to induce graft rejection. Genetic analysis of the rearranged VH genes from 10 cDNA clones demonstrated that the Ig µ (n = 5) and γ (n = 5) chain clones used the same family of VH genes (VHHAR family) to encode their antibody binding activity. The majority (80%) of the IgM clones were present in their original germline configuration. In contrast, the nucleotide sequences from IgG clones manifested an increase in the numbers of replacement mutations in the CDR region of the Ig heavy chain genes, providing evidence for a potential role for somatic mutation in the maturation of IgG xenoantibody responses as the humoral response matures with time post‐transplantation.

Identification and characterization of monoclonal antibodies that partially block human natural antibody binding to pig endothelial cell xenoantigens

Abstract: The shortage of human donors for clinical transplantation has led to a serious consideration of the use of non‐human species as organ donors. The major barrier to the clinical use of xenografts from species such as the pig in human transplantation has been the aggressive nature of the immune‐mediated rejection of the graft. We have recently identified the molecular weights of several endothelial cell surface proteins that may be targets of human antibody‐mediated responses to pig aortic endothelial cells (PAEC). In this series of experiments, we produced a panel of rat monoclonal antibodies (Mabs) to PAEC in an effort to identify Mabs that detect pig xenoantigens. Mabs were selected based on flow cytometric binding to PAEC, pig platelets, and various pig cell lines, including a pig kidney cell line (LLC‐PK1) reported to react with human natural antibodies (HNA). Eleven of the eighty‐three antibodies produced were cytotoxic for PAEC. Six of the cytotoxic clones recognized a 44 kDa protein and two of the clones recognized a 115 kDa protein expressed on the surface of PAEC. Since PAEC target antigens recognized by human natural antibodies include both 115 and 44 kDa antigens, these Mab clones were selected for further study. Several distinct patterns of tissue reactivity were demonstrated within this group of antibodies by immunohistochemical analysis; however all monoclonal antibodies were highly reactive with endothelial cells in all tissues examined. Two monoclonal antibodies recognizing antigens that are highly expressed on pig endothelial cells (92–98%) and pig platelets (74–92%), but moderately expressed on pig splenocytes (33–38%), were capable of reproducibly blocking 48–53% of human IgM binding to pig endothelial cells when analyzed with flow cytometry. This data suggests that these Mabs may recognize epitopes of potential significance in the human‐to‐pig xenograft reaction.