BACKGROUND:Treatment of naive CD4+ T cells in vitro with transforming growth factor-beta (TGF-beta) or TGF-beta/interleukin-2 (IL-2), combined with stimulation in a mixed lymphoid culture (MLC), has been shown to generate CD4+ CD25+ regulatory T cells. However, little is known about the effect of these regulatory T cells on cardiac allograft survival in vivo.METHODS:CD4+ CD25+ T cells were generated from Lewis (LEW) rat spleen through a primary MLC with TGF-beta (10 ng/ml) or TGF-beta/IL-2 (10 U/ml). The effect of adoptive transfer of the CD4+ CD25+ T cells (5.0 x 10(7)) was evaluated using an animal model of ACI rat cardiac allograft survival in LEW recipients.RESULTS:The MLC with TGF-beta or TGF-beta/IL-2 generated CD4+ CD25+ regulatory T cells, which suppressed the cytotoxic activity of LEW spleen T cells against irradiated ACI spleen cells in vitro. Adoptive transfer of the CD4+ CD25+ regulatory T cells intravenously to naive syngeneic recipients significantly prolonged the ACI cardiac allograft survival (N = 6, 13.5 +/- 3.4 days) compared with the control group (N = 6, 5.0 +/- 0.6 days).CONCLUSIONS:Intravenous administration of CD4+ CD25+ regulatory T cells, successfully generated by TGF-beta/IL-2 treatment, had a significant effect on cardiac allograft survival in this rat model. Adoptive transfer of regulatory T cells may represent a novel approach for preventing allograft rejection.

Background. Allograft fibrosis is a prominent feature of chronic rejection. Although intragraft fibroblasts contribute to this process, their origin and exact role remain poorly understood.Methods. Using a rat model of chronic rejection, LEW to F344, cardiac fibroblasts were isolated at the point of rejection and examined in a collagen gel contraction assay to measure fibroblast activation. The allograft microenvironment was examined using immunohistochemistry for fibrogenic markers (transforming growth factor [TGF]-beta, platelet-derived growth factor [PDGF], tissue plasminogen activator [TPA], plasminogen activator inhibitor [PAI]-1, matrix metalloproteinase [MMP]-2, and tissue inhibitor of matrix metalloproteinase [TIMP]-2). The origin of intragraft fibroblasts was studied using female to male allografts followed by polymerase chain reaction [PCR] and in situ hybridization for the male sry gene.Results. The cardiac fibroblasts isolated from allografts with chronic rejection exhibited higher gel contractibility (50.9% +/- 6.1% and 68.2% +/- 3.8% at 4 and 24 hr) compared with naive cardiac fibroblasts (30.7% +/- 3.5% and 55.3% +/- 6.6% at 4 and 24 hr; P < 0.05 and < 0.05, respectively). Immunostaining for TGF-beta, PDGF, TPA, PAI-1, AMP-2 and TIMP-2 was observed in all allografts at the time of rejection. In situ hybridization demonstrated the presence of sry positive cells in female allografts rejected by male recipients. Sixty-five percent of fibroblast colonies (55 of 85) isolated from female heart allografts expressed the male sry gene.Conclusion. Cardiac fibroblasts are activated and exist in a profibrogenic microenvironment in allografts undergoing chronic rejection. A substantial proportion of intragraft fibroblasts are recruited from allograft recipients in this experimental model of chronic cardiac allograft rejection.

and TCR-tg mice (n 6) do not acutely reject bm12 hearts (graft survival 33-100 days). However, TCR-tg mice primed with bm12 skin grafts acutely reject bm12 heart grafts (MST 22 days, n 6). Interestingly, TCR-tg recipients of bm12 heart grafts fail to develop chronic rejection compared with B6 recipients. We developed a model to visualize dynamics of CD4 T cell mediated direct allograft rejection and to investigate mechanisms underlying development of transplant tolerance. B6 nude mice were adoptively transferred with 15x10 TCR-tg spleen cells and transplanted with bm12 skin grafts. Expansion of allospecific TCR-tg CD4 T cells was observed primarily in the draining lymph nodes at early time points. Alloantigen-driven TCR-tg CD4 T cell expansion increased 7 fold over homeostatic expansion, peaked 14 days after transplantation, and declined thereafter. TCR-tg CD4 T cells exhibited upregulation of early activation markers, expression of effector/memory markers, and IFNproduction mirroring the kinetics of clonal expansion. We investigated the role of B7-CD28 and B7h-ICOS costimulatory blockade in MHC class II alloreactive CD4 T cell responses using CTLA4Ig and anti-B7h mAb. CTLA4Ig treated recipients exhibited a drastic reduction in TCR-tg CD4 T cell expansion. TCR-tg CD4 T cells also retained a naive phenotype. In contrast, anti-B7h mAb had no effect on expansion or activation of TCR-tg CD4 T cells. CTLA4Ig reduced IFNproduction by 80%, anti-B7h mAb by 20%. Our results represent the first model of MHC class II alloreactive CD4 T cell activation in vivo. It provides a powerful tool for tracking CD4 T cell mediated alloimmune responses and investigating mechanisms of tolerance induction in vivo.

Mesenchymal stem cells (MSC) are pluripotent progenitors for a variety of cell types, including fibroblasts and myocytes. MSC have been shown to participate in tissue repair of various organs following injury. However, their contribution to tissue repair of allografts during the development of chronic rejection has not been examined. We have recently isolated rat MSC from circulation using an aortic pouch allograft as a trapping device. The plasticity of these cells was examined in differentiation cultures. One of the resulting MSC lines was immortalized and transduced to express a marker lacz gene. The lacz -labeled cells were then transferred to F344 rats bearing LEW cardiac allografts for evidence of their homing to allograft and bone marrow, and their contribution to graft tissue repair. Our results showed that the MSC isolated from circulation had multi-potential for differentiation in culture, developing into various lineages including osteoblasts, lipocytes, chondrocytes, myotubes and fibroblasts. Intravenous engraftment of the lacz-labeled cells into recipients of heart transplant resulted in migration of the β-gal+ cells into the lesions of chronic rejection in the cardiac grafts, and homing of the cells to the bone marrow. The majority of β-gal+ cells present in the allografts exhibited fibroblast phenotypes and a small number of the cells expressed desmin, indicative of myocyte differentiation. Some of the lacz+ cells recovered from heart allografts expressed αSMA or desmin protein, indicating site-specific differentiation of the engrafted MSC toward myofibroblast and myocyte, respectively. We conclude that MSC are attracted to heart allograft to actively participate in tissue repair during chronic rejection. Given the robust MSC migration, the inhibition of MSC differentiation toward fibroblast progeny and induction toward the myocyte lineage may serve as a new strategy for treatment of chronic rejection and allograft tissue repair.

Non-MHC alloantibodies may play a pathogenic role in chronic rejection but remain poorly characterized. This study investigated the kinetics of antidonor antibody production and the injury mechanism in a LEW-to-F344 rat model of cardiac transplantation, in which donor and recipient strains differ for non-MHC alloantigens. All the F344 recipients of LEW allografts produced antidonor IgG antibodies reactive with LEW endothelial cells (EC). A subgroup of recipients that rejected their grafts in 30-60 days exhibited markedly higher levels of anti-donor IgG antibodies (n=6, MFI:23.85±2.7) prior to graft rejection when compared to those with long-surviving allografts (n=4, MFI:11.23±0.81; P=0.00058). F344-anti-LEW sera were capable of inducing lesions of chronic rejection when passively transferred in an antibody-free heart graft model in which immune non-responsiveness of F344 rats to LEW alloantigens was induced by intrathymic inoculation of LEW lymphocytes. Five of six cardiac allografts exposed to the antisera were rejected in 2-5 weeks after passive transfer and developed severe chronic lesions while control allografts (N=3) treated with normal rat sera survived for > 90 days (p=0.015). IgG antibodies purified from anti-LEW sera exhibited complement-dependent cytotoxicity against LEW EC in culture. The EC displayed early (Annexin V+) and late (TUNEL+) evidence for programmed cell death examined with flowcytometry. Western blot analysis of poly (ADP-ribose) polymerase (PARP) cleavage showed that an 116 kDa intact form of PARP was degraded into 85 kDa and 25 kDa fragments in lysates of the ECs treated with the IgG, indicating an apoptosis-associated caspase activity. In addition, in situ TUNEL experiment demonstrated that vascular EC apoptosis was consistently present inall the heart allografts examined. We conclude that non-MHC alloantibodies are pathogenic and capable of causing chronic graft injury via an antibody-induced cell apoptosis mechanism. The results emphasize the importance of non-MHC antibodies as a common predisposing factor in the development of chronic rejection.

Backgound: Non-major histocompatibility complex (non-MHC) alloantibodies may play a pathogenic role in chronic rejection but remain poorly characterized.Methods: The kinetics of alloantibody production and the mechanism by which non-MHC alloantibodies cause graft injury were investigated in a Lewis-to-Fischer 344 (LEW-to-F344) rat model of cardiac transplantation.Results: Flow cytometry detected that all the F344 recipients of LEW allografts produced anti-donor immunoglobulin G (IgG) antibodies reactive with LEW lymphocytes and endothelial cells. A sub-group of recipients that rejected their g rafts in 30 to 60 days exhibited markedly increased levels of anti-donor IgG antibodies (n = 6, mean fluorescence intensity [MFI]:23.85 +/- 2.7) than recipients with long-surviving allografts (n = 4, MFI:11.23 +/- 0.81; p = 0.00058). Passive transfer of anti-donor sera induced chronic rejection of LEW heart allografts in an immune non-responsiveness model of F344 rats induced by intrathymic inoculation of donor-specific lymphocytes. Immunoglobulin G antibodies purified from the anti-LEW sera exhibited complement-dependent cytotoxicity, against LEW vascular endothelial cells in flow-cytometric cytotoxicity assay. The targeted endothelial cells displayed early (annexin V+) and late (TUNEL+) evidence for programmed cell death. Western blot analysis of poly (ADPribose) polymerase (PARP) demonstrated that the 25-kD PARP-cleavage fragment was present at the lysates of the vascular endothelial cells treated with anti-donor IgG antibodies, indicating apoptosis-associated caspase activity in these cells. In situ teminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining demonstrated that vascular endothelial cell apoptosis was consistently present in all LEW heart allografts with chronic rejection.Conclusions: Non-MHC alloantibodies are pathogenic and capable of causing chronic graft injury through an antibody-induced cell apoptosis mechanism. The results emphasize the importance of non-MHC antibodies as a common predisposing factor in the development of chronic rejection.

Background Natural antibodies that react with galactose-alpha (1,3)galactose [gal alpha (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 gal alpha (1,3)gal antigen. The Gal knockout (Gal o/o) mouse, produced by homologous disruption of the alpha1,3GT gene, spontaneously makes anti-gal alpha (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 gal alpha (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 gal alpha (1,3)gal carbohydrates, The V-H and V-K genes of these hybridomas were cloned, sequenced, and analyzed.Results. The mAbs showed distinct patterns of antibody binding to gal alpha (1,3)gal antigens, The V-H 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 V-H germline gene (V(H)441). Comparison of the mouse gene V(H)441 to the human gene IGWV3-11, a gene that encodes antibody activity to gal alpha (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 V, genes.Conclusions. These results demonstrate the similarity of the Gal o/o mice and humans in their immune response to gal alpha (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.

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.

Wu, Gordon D.; Jin, Yang-Sun; Swenssion, Joyce; Starnes, Vaughn; Cramer, Donald V. Author Information

Wu, Gordon D.1; Gochi, Eiji2; Swensson, Joyce1; Starnes, Vauhn1; Cramer, Donald V.1 Author Information

446 The severe shortage of allogeneic organs available for human transplantation has led to the consideration of the use of pigs as organ donors. Human preformed and induced antibody responses to pig xenografts, however, present a major barrier to the successful use of pig organs for human transplantation. We have recently identified the IgVH genes responsible for encoding IgM xenoantibody responses in humans exposed to pig tissues in a bioartificial liver support device. These responses are encoded by Ig genes that are members of the VH3 family and are restricted to genes encoded by the IgHV3-11 and IGHV3-74 germline progenitors. Induced xenoantibody responses in human patients at 10 days post BAL exposure demonstrate a clonal expansion of a VH gene with a unique VDJ gene configuration. We have recently demonstrated that this clonally expanded gene encodes a functional antibody that reacts with the α-gal epitope. In this report we have extended our analysis of the host response to the Ig genes used to encode IgG xenoantibodies. We have generated IgG gene libraries established from lymphocytes of patients at days 0 and 22 following treatment with a bioartificial liver containing pig hepatocytes and endothelial cells. cDNA libraries representing the VH3 family were screened by colony filter hybridization to identify increases in the frequency of expression of specific VH genes encoding α-gal antibodies. Immunoglobulin genes derived from the IGHV3-11 germline progenitor demonstrate an increase in frequency of expression from 2.9% at day 0 to 20% at day 22. A CDR3 specific probe was used to identify an IgM to IgG isotype switch during the maturation of the antibody response. A unique, clonally expanded IGHV3-11 gene identified in IgM clones at day 10 represent the majority of IgG clones expressed at high levels at day 22. The identify of this clone was confirmed by DNA sequencing. IgVH genes encoded by the IGHV3-74 family demonstrate an increase in expression from 0.8% at day 0 to 5.5% at day 22. Genes related to V3-7 germline progenitors do not contribute to the xenoantibody response. These results demonstrate that the xenoantibody response in humans is encoded by IgVH genes restricted to IGHV3-11 and IGHV3-74 germline families. A specific xenoantibody reactive with the α-gal epitope undergoes a clonal expansion and an isotype switch during the maturation of the xenoantibody response. The restricted nature of the humoral response to pig tissues is of potential importance for the development of therapeutic strategies to prevent xenograft rejection.

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.

5 The target antigens for xenograft reaction appear to share many structural features for different species combinations. They are composed primarily of complex carbohydrate epitopes and in each case, the carbohydrate composition of the xenoantigens is similar to the glycosylated proteins and lipids in the cell walls of infectious agents, many of which are targets for natural antibodies that serve as an immune mechanism for preventing infection. The broad pattern of reactivity exhibited by xenoantibodies may be the results of their use of Ig V genes in their original germline configuration to provide a rapid response to a wide variety of common infectious agents present in the environment. In this report, we have examined the use of VH genes by xenoantibodies and compared the Ig VH gene structures of xenoantibodies in different species for similarities in the structure of their combination sites. Ig μ chain or γ chain cDNA libraries specific for the rearranged IgVH genes were constructed from lymphocytes derived from the following species: (1) rat recipients of hamster heart xenografts; (2) mouse-anti-gal hybridomas from Gal o/o mice; and (3) human patients responding to pig cells. The VH-D-JH regions of these cDNA clones, including 35 rat-anti-hamster clones. 4 mouse-anti-gal clones and 64 clones for human-anti-pig xenoantibodies, were sequenced and their amino acid sequences within the immunoglobulin combining sites analyzed. Examination of the canonical binding site structure demonstrated that 96% (24/25) of the rat Ig clones, 100% (4/4) of the mouse clones and 100% (64/64) of the human clones displayed a similar pattern of conserved combination site configuration. The amino acid residue patterns on the first hypervariable region (H1) of the VH segments exhibit one (group 1) of the three different structural groups. Similarly, the second hypervariable regions (H2) has one (group 3) of five different patterns. Furthermore, 90% of the clones were characterized by sharing a single amino acid (glycine) in position 54 within the CDR2 segment, a position predicated to a key antigenic contact site. A literature search revealed that a variety of antibodies against carbohydrates also exhibit the 1-3 binding site patterns. Our data, therefore, by demonstrating a profile of preferential utilization of the 1-3 combination site configuration by different xenoantibodies derived from various phylogenetically distantly related species, including rodents and humans, suggest that the 1-3 binding site structure is highly conserved during evolution and might have important role in antibody protection against infectious agents and xenografts.

860 The use of antibody engineering to manipulate and reshape antibodies has led to the production of antibodies with the ability to alter the course of immune responses by functioning to bind and inactivate molecules, including glycoproteins, at both intracellular and extracellular levels. Single chain antibodies have been demonstrated to have a variety of clinical applications at the therapeutic level. In the xenograft model, the rejection of pig organs transplanted into humans is mediated by xenoantibodies that react with the α-gal epitope. Human patients exposed to pig cells mount a xenoantibody response that is encoded by IgVH genes derived from the IGHV3-11 germline progenitor. We have recently used the colony filter hybridization technique to demonstrate a significant increase in the frequency of expression of genes with a specific VDJ gene configuration in IgM and IgG cDNA libraries prepared from the peripheral blood of patients exposed to pig cells. The restricted nature of this response strongly suggests that a specific and targeted manipulation designed to alter this xenoantibody response has the potential to prolong xenograft survival. We therefore cloned the IgHV3-11 gene encoding this antibody along with a light chain variable region into a phagemid vector (pHEN2) using an overlap extension PCR technique. The introduction of an amber mutation between the antibody genes and gIII allows soluble scFv fragments to be produced directly using the technique of induction with IPTG in a nonsuppressor E.coli strain. The soluble scFv fragments were purified and screened for binding to the α-gal epitope in an ELISA assay using bovine thyroglobulin and mouse laminin as antigenic targets. We constructed two clones producing single chain antibodies encoded by IGHV3-11 germline progenitors. Both clones demonstrated strong reactivity for the α-gal epitope expressed on both bovine thryroglobulin and mouse laminin. The reactivity of human single chain xenoantibodies to single stranded DNA and human thyroglobulin in an ELISA assay was used to determine whether human xenoreactive antibodies are polyreactive or monospecific and to address whether these clones demonstrated autoantibody activity. One clone demonstrated reactivity with the α-gal epitope in the absence of autoantibody activity and one clone demonstrated both autoantibody and α-gal reactivity. DNA sequencing will be used to address the role of specific amino acid substitutions in altering the specificity of the xenoantibody response.

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.