Pretransplant injection of allograft recipients with donor blood or lymphocytes permits allograft tolerance without the presence of persistent donor microchimerism

Donor-recipient microchimerism has recently been suggested to play a critical role in the induction and maintenance of allograft tolerance. In this study we sought evidence for this hypothesis using the LEW-to-ACI cardiac allograft as a model system. Donor-specific tolerance to cardiac allografts was induced by intravenous or intraportal injection of graft recipients with donor peripheral blood, T cells, or B cells 7 days before transplantation. All the graft recipients injected with donor antigens accepted donor heart grafts indefinitely when compared with control recipients that rejected donor allografts in 12 days. Long-term graft survivors rejected third-party BN heart allografts in 14 days without an adverse effect on the survival of the first LEW heart allografts, demonstrating the specificity of the tolerance. Tissue lysates prepared from heart, kidney, liver, bone marrow, thymus, lymph nodes, and spleen of tolerant (>120 days) graft recipients were analyzed for the presence of donor DNA using LEW T cell receptor C beta gene-specific primers for polymerase chain reaction that detects donor DNA at > or = 1:10,000 dilution. Donor DNA was detected in 77% of tolerant graft recipients. Chimeric recipients showed variations in the levels and presence of donor DNA in different tissues. The status of donor microchimerism, with respect to its presence and tissue distribution, was dependent upon the donor cell type and route of injection used for the induction of tolerance. Intraportal injection of the graft recipients with donor peripheral blood resulted in the highest degree of chimerism, whereas intravenous injection with donor B cells did not induce detectable microchimerism in this group of recipients. These data clearly demonstrate that the presence of microchimerism is common following administration of donor cells, but that its presence is not an absolute requirement for the long-term survival of allografts.     

Primary immunodeficiency diseases at Red Cross War Memorial Children''s Hospital

BACKGROUND: Normally, a Buffalo (BUF) recipient (RT1b) rejects a heterotopically transplanted Lewis (LEW) heart (RT1l) drained into the portal vein (PV) within 14 days. However, the addition of PV administration of 25x10(6) ultraviolet B (UVB)-treated LEW spleen cells (SC) to BUF recipients 7 days before cardiac transplantation results in 70% long-term allograft survival. METHODS: In this study, we used gadolinium chloride (GdCl3) (7 mg/kg/day) to selectively block the phagocytosis of recipient hepatic Kupffer cells before PV injection of UVB-treated donor SC to examine the mechanism of tolerance induction, as measured by in vitro analysis of mixed lymphocyte culture (MLC), T cell cytotoxicity, T helper cell precursors (pTH), and cytotoxic T cell precursors (pCTL) by limiting dilution analysis. RESULTS: A BUF recipient that received untreated or gamma-irradiated LEW SC intraportally reacted to in vitro stimulation by LEW alloantigen with increased MLC proliferation, T cell cytotoxicity, pTH and pCTL frequencies, and interleukin-2 production. In contrast, SC from BUF that received UVB-treated LEW SC were hyporesponsive on MLC stimulation by donor LEW alloantigen and exhibited markedly reduced cytotoxicity, pTH and pCTL frequency, and interleukin-2 production. However, normal in vitro responsiveness resulted with stimulation by third-party Brown-Norway (RT1n) SC, thus indicating that the systemic hyporesponsiveness was specific for the UVB donor alloantigen given PV. On the other hand, GdCl3 given by intravenous injection daily for 3 days before PV alloantigen blocked the induction of in vitro hyporesponsiveness. CONCLUSION: Therefore, prevention of alloantigen sequestration by GdCl3 inhibition of hepatic Kupffer cell phagocytosis was pivotal in preventing the development of portal venous tolerance.     

Isolation and characterization of murine clonogenic osteoclast progenitors by cell surface phenotype analysis

MHC class I molecules bind short peptides for presentation to CD8+ T cells. The determination of the three-dimensional structure of various MHC class I complexes has revealed that both ends of the peptide binding site are composed of polar residues conserved among all human and murine MHC class I sequences, which act to lock the ends of the peptide into the groove. In the rat, however, differences in these important residues occur, suggesting the possibility that certain rat MHC class I molecules may be able to bind and present longer peptides. Here we have studied the peptide length preferences of two rat MHC class Ia molecules expressed in the TAP2-deficient mouse cell line RMA-S: RT1-A1c, which carries unusual key residues at both ends of the groove, and RT1.Aa which carries the canonical residues. Temperature-dependent peptide stabilization assays were performed using synthetic random peptide libraries of different lengths (7-15 amino acids) and successful stabilization was determined by FACS analysis. Results for two naturally expressed mouse MHC class I molecules revealed different length preferences (H2-Kb, 8-13-mer and H2-Db, 9-15-mer peptides). The rat MHC class Ia molecule, RT1-Aa, revealed a preference for 9-15-mer peptides, whereas RT1-A1c showed a more stringent preference for 9-12-mer peptides, thereby ruling out the hypothesis that unusual residues in rat MHC molecules allow binding of longer peptides.     

Interstrain variability of autoimmune encephalomyelitis in rats: multiple encephalitogenic myelin basic protein epitopes for DA rats

We investigated T cell epitopes of guinea pig myelin basic protein (MBP) that induce experimental autoimmune encephalomyelitis (EAE) in DA rats, using synthetic peptides that correspond to regions of the guinea pig MBP molecule that are homologous to rat MBP. Four peptides were encephalitogenic when tested in DA rats. MBP63-81, which partially overlaps the dominant encephalitogenic MBP epitope for Lewis (LEW) rats, caused severe EAE in the DA strain but did not elicit EAE in LEW rats. MBP66-81 and MBP63-76 were also encephalitogenic for DA but not LEW rats. MBP79-99 also induced EAE in DA rats, although MBP87-99, the minor encephalitogenic LEW epitope, was inactive. This indicates that part of the 79-86 sequence is necessary for encephalitogenic activity in the DA strain. MBP101-120, and MBP142-167 were also encephalitogenic for DA rats. T cells from DA rats immunized with intact MBP proliferated in response to the whole protein and to MBP79-99, but were not stimulated to a significant extent by the other encephalitogenic peptides, suggesting that these may represent cryptic or subdominant epitopes. However, MBP63-81-specific T cell lines could be isolated by repeated restimulation with peptide, indicating that the peptide-specific T cells were present in DA rats at low frequency.     

Engraftment characteristics of peripheral blood stem cells mobilised with cyclophosphamide and the delayed addition of G-CSF

Avridine is a potent synthetic adjuvant that can induce arthritis is most rat strains. The clinical appearance and histopathology of avridine-induced arthritis show great similarity to other arthritis models such as collagen-induced arthritis. In LEW and DA rats the avridine-induced arthritis is severe and long lasting. To investigate a possible genetic influence on the disease we compared LEW, DA and E3 rats, which are of different genetic origins, for their ability to develop arthritis after injection of a low dose of avridine (1.5 mg/rat). The E3 rat was shown to be resistant, whereas all of the DA rats developed arthritis. Recombinant inbred strains derived from DA and E3 parentals varied in susceptibility to avridine. Only strains sharing RT1av1 with DA developed arthritis, indicating a role for the MHC genes. The MHC association was further analysed in a series of Lewis congenic strains using the 1.5 mg avridine dose. All strains developed arthritis. LEW.1C and LEW.1W developed only acute arthritis, whereas LEW.1A, LEW, LEW.1D, LEW.1N and LEW.1F developed chronic arthritis. In particular, the LEW.1F rats developed a chronic severe arthritis of high incidence. The chronic arthritis showed an active, erosive joint inflammation several months after induction. Nude rats are resistant to avridine-induced arthritis, indicating a T cell dependence of the disease which supports the importance of MHC. However, non-MHC genes are also crucial to arthritis development. Recombinants between DA and E3, sharing RT1av1 with DA, showed either a lower incidence or a lower severity of disease than the DA rats. The E3 rat and the recombinants with RT1u were completely resistant, whereas LEW.1W, also RT1u, were highly susceptible.     

Importance of T cells to accelerated rejection and acceptance of renal allografts in sensitized rat recipients

BACKGROUND: Sensitized recipients often experience fulminant allograft loss by yet ill-defined cellular and/or humoral immune mechanisms. In this study, we analyzed the contribution of cellular elements, in particular T cells, to the accelerated rejection of renal allografts in sensitized rats. METHODS AND RESULTS: LEW rats sensitized with BN skin grafts died of uremia in 3.3+/-0.9 days after transplantation of a BN kidney, similarly to bilaterally nephrectomized animals. Adoptive transfer of 10(6) graft-infiltrating mononuclear cells as well as their CD25+ subset into otherwise normal LEW recipients accelerated rejection of BN test cardiac allografts (5.4+/-0.5 days to 6.6+/-0.4 days vs. 7.8+/-0.8 days in controls, P<0.0007), while the CD25- population was ineffective (8.0+/-0.6 days, NS). Furthermore, alpha/beta-T-cell receptor (TCR)-targeted therapy with R73 monoclonal antibody abrogated accelerated rejection, and produced long-term survival in sensitized animals treated before kidney engraftment (day -7 to day -1). Long-term survival was associated with an up-regulation of intragraft interleukin-4 and interleukin-10 expression in conjunction with depressed Th-1-type cytokines. In addition, alpha/beta-TCR-targeted therapy even in low subtherapeutic dose decreased IgM alloantibody levels, and prevented the switch from IgM to IgG alloantibody response. CONCLUSIONS: This is the first report that documents the striking efficacy of alpha/beta-TCR-targeted therapy in sensitized rat renal transplant recipients. The results provide evidence for a critical role of T cells for both accelerated rejection and long-term graft survival. Up-regulation of Th2-type cytokine profile may, at least in part, contribute to the acquisition of immune unresponsiveness after alpha/beta-TCR-targeted therapy in this well-defined rat renal transplant model.     

Induction of allograft nonresponsiveness after intrathymic inoculation with donor class I allopeptides. II. Evidence for persistent chronic rejection despite high levels of donor microchimerism

We have recently demonstrated that three synthetic peptides corresponding to the donor class I RT1.Aa molecule induce long-term survival of cardiac allografts in the PVG.R8-to-PVG.1U rat strain combination (disparate for one isolated class I, RT1.A, molecule) when presented to the recipient immune system in the thymus. Long-term graft survivors had measurable levels of donor-reactive alloantibodies in their serum. In this study, we examined long-term allografts for the presence of chronic rejection and donor microchimerism to assess whether this regimen of immune modulation establishes true tolerance and whether this tolerance is dependent upon the presence of donor-recipient microchimerism. Histological examination of long-term heart grafts (>100 days) demonstrated chronic rejection, including a mild degree of myocardial infiltration by mononuclear cells, mild to moderate myocardial fibrosis, and various vascular changes ranging from focal intimal thickening to total vascular lumen blockade due to smooth muscle cell proliferation. In contrast, long-term syngeneic hearts transplanted under similar experimental conditions lacked these pathological manifestations. Donor microchimerism was analyzed using the polymerase chain reaction with a pair of oligonucleotides specific for the donor class I RT1.Aa gene and genomic DNA harvested from various tissues from graft recipients. We detected high levels of donor microchimerism in the heart, kidney, liver, skin, bone marrow, thymus, and lymph nodes of long-term graft recipients. Donor microchimerism was also detected in unmanipulated control graft recipients at rejection (7 days) and in intrathymically manipulated recipients that rejected allografts in a delayed fashion (12-82 days). These data clearly demonstrate that intrathymic inoculation of donor class I allopeptides induces long-term graft survival but does not prevent chronic rejection. Allograft rejection occurred despite high levels of donor microchimerism, providing direct evidence that donor-recipient microchimerism is not sufficient for the prevention of acute or chronic rejection in this model.     

Antibody- and cell-mediated immune responses of Actinobacillus pleuropneumoniae-infected and bacterin-vaccinated pigs

The number involved in and the rate of migration of donor leucocytes into the following recipient organs (spleen, thymus, bone marrow, lung and mesenteric lymph nodes) were measured in two rat models of orthotopic liver transplantation (OLT) using donor-specific MHC class I antibodies. The first OLT model is one that does not require immunosuppression in order to achieve tolerance and involved the transplantation of DA (MHC haplotype, RT1a) livers into PVG (RT1c) recipients. The second model was one that required a 7-day (10 mg/kg) treatment with cyclosporin A (CsA) to achieve tolerance and used DA donors into Lewis (RT1(1)) recipients. Recipient organs were biopsied on days 3, 20 and 87 following OLT and donor leucocyte migration was quantified by immunohistochemistry and computer densitometry of immunoblots of detergent-solublized tissues in order to resolve both membrane-bound and soluble donor MHC class I antigen. In a separate experiment, spleen biopsies were taken following OLT on days 3 and 15 from the naturally tolerizing OLT model (DA into PVG), treated with and without CsA for 7 days and compared with the (DA into Lewis) model. The initial rate of leucocyte migration between days 3 and 21 following OLT was found to be the most rapid into the spleen, followed by the bone marrow and mesenteric lymph nodes in the naturally tolerant (DA into PVG) model when compared with the (DA into Lewis) model. The number of donor leucocytes in the spleen and mesenteric lymph nodes in both models was, however, approximately the same by 87 days. No real difference in the rate of leucocyte migration was seen in the thymus or the lung for both transplant models over the time course assayed. CsA was found to lower the rate of donor leucocyte migration only over the period it was administered. The rate of donor leucocyte migration into the spleen was still much lower 15 days after OLT in the (DA into Lewis) model compared with the (DA into PVG) model treated with and without CsA. Thus the differences in the rate of donor leucocyte migration into the spleen, bone marrow and mesenteric lymph nodes immediately following OLT may offer an explanation as to why the (DA into PVG) combination is able to accept a transplanted liver without immunosuppressive therapy.     

MHC class I molecules compete in the endoplasmic reticulum for access to transporter associated with antigen processing

We have used the functionally distinct TAP alleles of the rat in cellular transfectants as tools to investigate how newly formed rat class I (RT1.A) molecules with distinct peptide requirements gain access to suitable peptides in the endoplasmic reticulum (ER). Normal maturation of RT1.Aa depends on the presence in the ER of peptides with C-terminal arginine, while restrictive TAP-B allelic group transporters fail to transport such peptides. In this situation, RT1.Aa is retained in the ER. We show that this retention is accompanied by accumulation of RT1.Aa in the ER, partly associated with TAP and partly free. In such cells, access to TAP of a second allelic product, RT1.Au, which does not require C-terminal arginine peptides, is competitively inhibited by the build-up of RT1.Aa. Nevertheless, RT1.Au loads and matures normally. Introduction of a permissive TAP-A allele competent to transport C-terminal arginine peptides releases RT1.Aa from the ER and restores RT1.Au interaction with TAP. Both class I alleles associate indiscriminately with permissive and restrictive TAP alleles. The data support the view that interaction with TAP is not a prerequisite for peptide loading by class I molecules, so long as suitable peptides are available in the ER. They further show that TAP association of a class I molecule depends on a competitive balance in the ER defined by the extent to which the peptide requirements of other class I molecules present are satisfied and not only by the intrinsic strength of the interaction with TAP.     

Development of anti-tissue antibodies in the rat liver transplant model

BACKGROUND: The aim of this study was to analyze the humoral immune response associated with orthotopic liver transplantation in the rat liver transplant model, and in particular to test the presence of anti-tissue antibodies. METHODS: Rearterialized liver transplantations were performed in the Dark Agouti (DA)-to-Lewis (LEW) and the LEW-to-DA rat strain combinations. Sera of recipients were analyzed by immunofluorescence (on DA and LEW organ sections) and by western blotting (with DA and LEW liver proteins). RESULTS: We have shown that liver (but not heart or skin) recipients develop a humoral response against non-MHC tissue antigens as evidenced (1) by a pattern of staining comparable to that described in human patients harboring anti-smooth muscle antibodies and (2) by the presence of donor liver peptides recognized in the sera of the recipient by Western blotting. CONCLUSIONS: These experiments indicate that orthotopic transplantation of a nonacutely rejected liver allograft is associated with the development of a previously undescribed anti-tissue antibody response that seems to be neither organ nor MHC restricted.     

Tolerance to experimental cardiac allografts produced by neonatal intrathymic injection of donor cells

Intrathymic inoculation of allogeneic cells after systemic administration of antilymphocyte serum in adult experimental animals has produced donor-specific tolerance to cardiac allografts. We investigated whether thymic injection of allogeneic cells without antilymphocyte serum in neonatal Lewis rats (day 1 of life) with immature immune systems also produced tolerance to cardiac grafts. Intrathymic or intraperitoneal injection of 5 x 10(7) Lewis (control) or Lewis-Brown Norway (allogeneic) spleen cells in Lewis neonates was followed by heterotopic cardiac transplantation using Lewis, Lewis-Brown Norway, or Wistar Furth (third-party allograft) hearts at 6 to 8 weeks of age. Graft survival was prolonged with both intraperitoneal and intrathymic allogeneic cells. Recipients of cells by the intrathymic route had longer graft survival, and 2 of 5 animals achieved permanent graft acceptance (longer than 100 days). As expected, Lewis isografts survived indefinitely, whereas third-party Wistar Furth allografts were rejected in the usual time frame. Intrathymic introduction of allogeneic cells in a neonatal recipient with an immature immune system can produce donor-specific tolerance to a subsequent graft without the need for a systemic immunosuppression regimen, even transiently.     

Distinct mechanisms for the induction and maintenance of allograft tolerance with CTLA4-Fc treatment

A murine CTLA4/Fc gamma2a heavy chain (mCTLA4-Fc) chimeric fusion molecule was used in B6AF1 recipients of BALB/c pancreatic islet allografts to study the induction and maintenance of tolerance following inhibition of the CD28-B7 pathway for T cell activation. Donor-specific tolerance was achieved by administering 100 microg of mCTLA4-Fc on alternate days for 14 days (8 total doses) or a single 500 microg dose of mCTLA4-Fc on day 2 after transplant. Tolerance was mediated by long-lived peripheral lymphocytes and showed features of organ and alloantigen specificity. Whereas tolerance could not be established in allograft recipients receiving simultaneous mCTLA4-Fc and rIL-2, previously tolerant animals did not reject their grafts when given IL-2, suggesting that the induction and maintenance phases of tolerance were distinct and separate. The maintenance of donor-specific tolerance was an active immunologic process that was CD4+ T cell dependent and could be adoptively transferred to naive lymphocytes, but could not be explained by apoptosis or deletion of alloreactive T cells. Although an IL-2-sensitive mechanism such as anergy may contribute toward the induction of tolerance, its maintenance involves active suppression.     

Role of passenger leukocytes in allograft rejection: effect of depletion of donor alveolar macrophages on the local production of TNF-alpha, T helper 1/T helper 2 cytokines, IgG subclasses, and pathology in a rat model of lung transplantation

Acute lung allograft rejection is believed to be initiated by passenger leukocytes, such as alveolar macrophages (AM), in the donor organ, which release TNF-alpha, and present alloantigens to host lymphocytes, to up-regulated Th1 cellular and humoral immunity. However, the role of donor AM in local TNF-alpha synthesis, and their ability to induce local Th1 cellular and humoral immunity have not been evaluated. By depleting Brown Norway (BN, RT1n) rat lung allografts of AM before transplantation into Lewis rat (LEW, RT1(1)) recipients, the current study determined the role of donor AM in including the production of TNF-alpha, IFN-gamma (Th1 cytokine), IL-4 (Th2 cytokine), IgG subtypes, and rejection pathology in the allograft. The data show that compared with untreated BN allografts, pretransplant depletion of donor lung AM resulted in significantly less TNF-alpha, and IFN-gamma production in allograft bronchoalveolar lavage fluid with variable effects on local IL-4 production. Depletion of AM in the donor lung before transplantation affected the local production of several IgG subclasses. However, pretransplant depletion of donor AM had no effect on the development of the pathology of severe acute rejection. These data show that donor AM have a central role in the local synthesis of TNF-alpha and induce the production of IFN-gamma and IgG subtypes, locally, during acute lung allograft rejection. However, depletion of AM before transplantation does not prevent the development of severe acute rejection in BN rat lungs, transplanted into LEW recipients.     

A hexapeptide VTKFYF from C-terminal part of interleukin-1 receptor antagonist, an inhibitor of IL-1-IL-1 receptor interaction

In order to find the low-molecular-weight interleukin-1 (IL-1) inhibitors we synthesized a series of peptides, derived from three regions of interleukin-1 receptor antagonist (IL-1ra): N-terminal (residues 5-9), central (90-98) and C-terminal (143-148). The decision was based on the thorough analysis of structural and functional properties of IL-1 proteins and the resemblance of some fragments of IL-1ra to well-known immunomodulators, like thymopentin and tuftsin. The competition between our peptides and IL-1 was measured as the inhibition of IL-1 induced IL-2 production in LBRM/CTLL cell line system. The activity of tuftsin (TKPR), a peptide immunomodulator derived from IgG molecule, was also examined. All peptides presented some activity, although the most interesting results (when the range of activity and dose-dependence were taken into account) were obtained for tuftsin and peptide VTKFYF from the C-terminal part of IL-1ra, which is in agreement with the latest reports on the structure of IL-1ra-receptor complex.     

Rejection of cardiac allografts by T cells expressing a restricted repertoire of T-cell receptor V beta genes

We have recently shown that T cells infiltrating cardiac allografts early in graft rejection use a limited T-cell receptor (TCR) V beta repertoire. In this study we tested whether this limited repertoire of V beta genes is important for graft rejection. A cell line, AL2-L3, was established from LEW lymphocytes infiltrating ACI heart allografts 2 days after transplantation. This cell line is composed of CD4+ T cells that primarily recognize the class II RTI.B major histocompatibility complex (MHC) molecule expressed by the donor graft. This cell line precipitated acute rejection of donor hearts with a median survival time (MST) of 10.5 days following adoptive transfer to sublethally irradiated LEW recipients. This rate of graft rejection was significantly (P < 0.0007) accelerated when compared with a MST of 60 days for allografts in irradiated control recipients. The AL2-L3-mediated acceleration of graft rejection was donor specific as WF third-party heart allografts were rejected with a delayed tempo (MST = 28.5 days). The V beta repertoire of this cell line was primarily restricted to the expression of V beta 4, 15 and 19 genes. The nucleotide sequence analysis of the beta-chain cDNAs from this cell line demonstrated that the restricted use of the V gene repertoire was not shared with the N, D and J regions. A wide variety of CDR3 loops and J beta genes were used in association with selected V beta genes. These data provide evidence for the role a restricted repertoire of V beta genes plays in cardiac allograft rejection in this model. The restricted usage of the V beta repertoire in an early T-cell response to allografts may provide the opportunity to therapeutically disrupt the rejection reaction by targeting selected T-cell populations for elimination at the time of organ transplantation.     

Inhibition of experimental autoimmune myasthenia gravis by major histocompatibility complex class II competitor peptides results not only in a suppressed but also in an altered immune response

To assess the capacity of major histocompatibility complex (MHC) class II-binding competitor peptides in inhibiting antibody-mediated disease processes, we studied experimental autoimmune myasthenia gravis in Lewis rats. Experimental autoimmune myasthenia gravis, a disease model mediated by T cell-dependent autoantibodies against acetylcholine receptors, was induced by immunization with Torpedo californica acetylcholine receptor emulsified in complete Freund's adjuvant. The immunodominant acetylcholine receptor T cell epitope was recognized by T cells in the context of MHC class II RT1.B(L). The disease inhibitory capacity of RT1.B(L)-binding peptides not related to the acetylcholine receptor was determined upon co-immunization with Torpedo acetylcholine receptor. Co-immunization of peptide OVA323-339, a strong RT1.B(L)-binding competitor peptide, resulted in complete disease inhibition. Although, the priming of the anti-acetylcholine receptor T cell response was not fully inhibited, the kinetics of the response was changed. Moreover, besides a drastic reduction of the anti-Torpedo acetylcholine receptor antibody titers, a shift in isotype distribution was found. These findings indicate that antibody-mediated autoimmune processes can be suppressed by MHC class II competitor peptides. Furthermore, the administration of such peptides in vivo not only passively inhibits T cell activation, but also functionally alters the immune response.