A critical role for transforming growth factor-beta in donor transfusion-induced allograft tolerance.

Donor-specific (DST) or nonspecific blood transfusions administered before transplantation can enhance survival of vascularized allografts both in humans and animals but the immunological mechanisms of this effect remain unclear. We have analyzed the expression and the role of endogenous TGF-beta1 in a model of heart allograft tolerance, induced by pregraft DST in adult rats. We reported previously that this tolerance occurs despite a strong infiltration of leukocytes into the graft that are unable to produce both Th1- and Th2-related cytokines in vivo. Allografts from DST-treated rats express high levels of TGF-beta1 mRNA and active protein. This phenomenon is correlated with the rapid infiltration of leukocytes producing high amounts of TGF-beta1. TGF-beta1-producing cells are virtually absent among early infiltrating cells in rejected grafts but are found at a later time point. The induction of allograft tolerance in vivo is abrogated by administration of neutralizing anti-TGF-beta mAb. Moreover, overexpression of active TGF- beta1 in heart allografts using a recombinant adenovirus leads to prolonged graft survival in unmodified recipients. Taken together, our results identify TGF-beta as a critical cytokine involved in the suppression of allograft rejection induced by DST and suggest that TGF-beta-producing regulatory cells are also involved in allograft tolerance.

T cell receptor repertoire usage in allotransplantation: an overview.

Lymphocytes express antigen receptors that allow the immune system to specifically recognize antigens. In transplantation, T cells play a critical role in the rejection process, and different protocols inhibiting T cell-mediated alloreactivity efficiently achieve prolongation of allograft survival. T cells can interact with alloantigens by two ways, either by the "indirect" pathway that correspond to the physiological mechanism of T cell immune recognition, or through the "direct" pathway where they recognize alloantigens directly on the surface of donor cells. If some T cells are specifically activated in allorecognition, one should be able to indirectly detect this "selection" by analyzing the T cell receptor usage that could be biased and reflect the preferential amplification of alloreactive lymphocyte subsets. Nevertheless compared with disease states such as cancer or autoimmunity the T cell receptor repertoire is still largely uncharacterized. We review the current results available on T cell repertoire usage in transplantation studies involving humans or various animal models. The T cell receptor repertoire involved in transplantation (restricted or unrestricted) and the features potentially common to alloimmune responses will be discussed.

Graft-infiltrating T helper cells, CD45RC phenotype, and Th1/Th2-related cytokines in donor-specific transfusion-induced tolerance in adult rats.

Specific tolerance to LEW.1W (RT1u) heart allografts can be induced in adult LEW.1A (RT1a) rats by donor-specific blood transfusion (DST). We have previously shown that both rejected and tolerated grafts are heavily infiltrated by T lymphocytes, and that in both cases these T cells are capable of developing similar cytotoxic responses against donor cells in vitro; tolerance is therefore not due to the deletion of alloreactive T cells. At the same time, we found that the accumulation of IL-2 and IFN-gamma mRNA was decreased in tolerated grafts compared with rejected grafts. These results suggested that the induction of allograft tolerance in DST-treated animals could be mediated by anergy or suppression of graft-infiltrating Th1 cells. Although Th1 and Th2 clones have not yet been characterized in the rat, peripheral CD4+ rat T cells can be divided into two populations, based on their expression of the isoform RC of the CD45 molecule. Upon activation, CD45RChigh CD4+ T cells produce IL-2 and IFN-gamma and responsible for the induction of the graft-versus-host reaction, whereas CD45RClow CD4+ T cells produce IL-4 in vitro and provide B cell help. In the present study, we show that heart allografts from both DST-treated and untreated rats were infiltrated by equivalent numbers of leukocytes, of which CD4+ T cells also made up similar percentages. Among these CD4+ T cells, we observed that in allografts from DST-treated recipients the CD45RChigh population on day 5 was very significantly smaller (P = 0.004) than in the untreated group, while CD45RClow populations remained comparable. Moreover, using a new quantitative RT-PCR method, we found a dramatic reduction in the accumulation of IL-2, IFN-gamma, IL-10, IL-4, and IL-13 mRNA in hearts from DST-treated recipients compared with those of untreated recipients during the week following transplantation. These results show that in heart allografts from DST-treated recipients, despite phenotypic changes suggesting Th1 inhibition by Th2 imbalance, T helper function was inhibited as a whole, and that in vivo the phenotype CD4+ CD45RClow does not always correlate with Th2-related cytokine-producing cells.

Fas ligand, tumor necrosis factor-alpha expression, and apoptosis during allograft rejection and tolerance.

BACKGROUND: Cytotoxic T cells can induce target cell lysis and apoptosis by different pathways. The interactions of CD95 antigen (Fas) with its ligand (CD95L) and of tumor necrosis factor (TNF)-alpha with its receptor (TNF-R1) lead to apoptotic cell death. Recently, conflicting studies have been published concerning the expression and the role of CD95L in allograft rejection and tolerance. METHODS: In this study, the intragraft expression of CD95/CD95L and TNF-alpha and the frequency and distribution of apoptotic cells were compared in a model of heterotopic cardiac allograft in the rat in which recipients were either not treated (acute rejection) or pretreated with donor-specific blood transfusion (tolerant). RESULTS: In the acutely rejected allografts, a peak in the expression of CD95L and TNF-alpha and in the number of apoptotic cells was observed during the first week after transplantation; apoptotic cells were confined to graft-infiltrating cells. In the tolerated allografts, however, levels of graft-infiltrating cell apoptosis and CD95L and TNF-alpha expression during the same period of time were dramatically lower. The expression of Fas was constitutive and was not modulated during acute rejection or tolerance. CONCLUSION: This down-regulation of CD95L and TNF-alpha in allografts rendered tolerant by donor-specific transfusion suggests a role for apoptosis-inducing pathways in acute allograft rejection.

Prolongation of allogeneic heart graft survival in rats by administration of a peptide (a.a. 75-84) from the alpha 1 helix of the first domain of HLA-B7 01.

Allospecific T lymphocytes mediate graft rejection through specific, direct or indirect, recognition of processed determinants of foreign MHC class I molecules. Small synthetic peptides derived from highly conserved sequences of the alpha 1 helix of the first domain of certain MHC class I molecules have been shown to inhibit CTL responses in vitro and to prolong graft survival in rats when combined with subtherapeutic doses of cyclosporine. Here, we report that the survival of LEW.1W heart allografts was significantly prolonged when transplanted into congenic LEW.1A recipients treated only with a peptide corresponding to residues 75-84 of the human HLA-B7-01 molecule (B7.75-84) before transplantation. The experimental value for mean survival time (+/- SD) in untreated recipients was 13 +/- 6 days and in peptide-treated recipients was 42 +/- 27 days (P < 0.002). A total of 64% of treated recipients had a functioning graft at 30 days, while grafts were rejected in all rats belonging to the control group within this time. Within graft-infiltrating leukocytes (GIL) in B7.75-84-treated animals, the proportion of T cells was significantly lower and that of CD5-/TCR alpha beta-/CD16-/CD8+ and MHC class II+ cells concomitantly increased, as compared with nontreated animals. GIL from B7.75-84-treated animals also exhibited a dramatic decrease (approximately 70%) of allospecific and spontaneous (NK) cytotoxic activity, whereas their proliferation and IL-2 production were similar in both experimental groups. The IFN-gamma, IL-2, and IL-10 mRNA levels from GIL from peptide-treated recipients were similar to levels of controls, reflecting a state of activation of GIL. Perforin and granzyme A mRNA, the level of which may be modulated parallel to impaired cytotoxic functions, were at similar levels in both experimental groups. These data demonstrate that B7.75-84 significantly prolongs graft survival in LEW.1A rats when given as a single agent and suggests that a specifically decreased cytotoxic response (allospecific and spontaneous) plays a major role.

Cbl-mediated negative regulation of platelet-derived growth factor receptor-dependent cell proliferation. A critical role for Cbl tyrosine kinase-binding domain.

The Cbl proto-oncogene product has emerged as a novel negative regulator of receptor and non-receptor tyrosine kinases. Our previous observations that Cbl overexpression in NIH3T3 cells enhanced the ubiquitination and degradation of the platelet-derived growth factor receptor-alpha (PDGFRalpha) and that the expression of oncogenic Cbl mutants up-regulated the PDGFRalpha signaling machinery strongly suggested that Cbl negatively regulates PDGFRalpha signaling. Here, we show that, similar to PDGFRalpha, selective stimulation of PDGFRbeta induces Cbl phosphorylation, and its physical association with the receptor. Overexpression of wild type Cbl in NIH3T3 cells led to an enhancement of the ligand-dependent ubiquitination and subsequent degradation of the PDGFRbeta, as observed with PDGFRalpha. We show that Cbl-dependent negative regulation of PDGFRalpha and beta results in a reduction of PDGF-induced cell proliferation and protection against apoptosis. A point mutation (G306E) that inactivates the tyrosine kinase binding domain in the N-terminal transforming region of Cbl compromised the PDGF-inducible tyrosine phosphorylation of Cbl although this mutant could still associate with the PDGFR. More importantly, the G306E mutation abrogated the ability of Cbl to enhance the ligand-induced ubiquitination and degradation of the PDGFR and to inhibit the PDGF-dependent cell proliferation and protection from apoptosis. These results demonstrate that Cbl can negatively regulate PDGFR-dependent biological responses and that this function requires the conserved tyrosine kinase binding domain of Cbl.

Selection of T cell clones with restricted TCR-CDR3 lengths during in vitro and in vivo alloresponses.

In a model of heart allograft rejection in adult congeneic rats mismatched for both class I and class II MHC molecules, we analyzed the TCR beta chain repertoire of T cells infiltrating rejected allografts [graft-infiltrating T cells (GITC)]. Although all BV families were used by GITC, oligoclonal expansions reflected by an altered distribution of TCR beta chain CDR3 lengths were detected throughout the rejection process. Interestingly, expansions involving TCR beta chains with common length and BV usage were recurrently found within distinct individuals at late stages of rejection in vivo and after in vitro mixed lymphocyte culture between donor and naive recipient cells. Sequence analysis of the CDR3 regions within recurrent TCR beta chains comprising either BV2 or BV13 gene segments demonstrated a complete sequence identity between BV2-BJ2S3 junctions derived from GITC in all individuals tested and the presence of conserved amino acids at constrained CDR3 positions within GITC BV13+ junctions derived from most individuals. These results suggest the existence of several major alloantigens responsible for expansion of T cell clones bearing a 'public' beta chain rearrangement within rejected allografts. The demonstration that such clones are also expanded during in vitro mixed lymphocyte reactions provides an experimental approach which might allow molecular characterization of the above major alloantigens and their possible in vivo targeting.

Regulation of CD95 (APO-1/Fas) receptor and ligand expression by lipopolysaccharide and dexamethasone in parenchymal and nonparenchymal rat liver cells.

The effect of lipopolysaccharide (LPS) on the expression of CD95 (APO-1/Fas) receptor and ligand (CD95L) was studied in primary cultures of rat liver Kupffer cells (KCs), sinusoidal endothelial cells (SECs), and parenchymal cells (PCs) at the messenger RNA (mRNA) level and by means of immunocytochemistry. LPS treatment of KCs and SECs led to a three- to five-fold increase in CD95L mRNA levels within 6 hours, which declined thereafter. Within 24 hours, the number of KCs and SECs staining positive for CD95L strongly increased. After a lag phase of 12 hours after LPS addition, in both cell types the mRNA levels for the soluble CD95 isoform increased approximately 10-fold; however, the number of KCs and SECs staining positive for transmembrane CD95 remained low and did not significantly increase. Compared with nonparenchymal cells, CD95L mRNA levels in primary hepatocyte cultures were low in the absence and presence of LPS. On the other hand, functionally active CD95 expression markedly increased in response to LPS in these cells. Dexamethasone diminished the LPS-induced stimulation of CD95L expression in nonparenchymal cells but markedly stimulated CD95L expression in PCs. Apoptosis of PCs and thymic lymphocytes was stimulated by the addition of supernatants derived from LPS-treated KC or SEC cultures and was apparently mediated by CD95L as assessed by its sensitivity to inhibitors of the CD95-dependent apoptotic pathway in PCs. The data suggest a complex and timely coordinated interplay between the various liver cell populations with respect to LPS-induced activation of the apoptotic machinery with potential relevance for immunoregulation.

Donor-specific blood transfusion-induced tolerance in adult rats with a dominant TCR-Vbeta rearrangement in heart allografts.

Following allotransplantation, determinants encoded within the donor MHC are recognized by recipient T lymphocytes through their Ag receptor. In this study, we investigated the TCR Vbeta chain diversity of T cells infiltrating rejected and tolerated heart allografts in a model of donor-specific blood transfusion-induced tolerance in MHC-mismatched congeneic rats. The PCR-based method that we used allows the diversity of Vbeta chains at the complementarity-determining region 3 level to be analyzed quantitatively. Our results show that the Vbeta repertoire usage in graft-infiltrating T cells was characteristic and different in tolerated compared with rejected grafts, and differed in both cases from the normal distribution of the Vbeta repertoire. An expansion of lymphocytes showing a conserved Vbeta18-Dbetal-Jbeta2.7 gene rearrangement was found, from the first day after grafting onward, in graft-infiltrating cells from all tolerant animals. This clone accounted for as much as 5% of the whole Vbeta repertoire in tolerated hearts, as evidenced by RNase protection assay. In contrast, we demonstrated that, of lymphocytes infiltrating rejected grafts, those with a Vbeta18 chain were diverse, and that even though by day 5 the conserved Vbeta18-Dbeta1-Jbeta2.7 rearrangement was detectable, lymphocytes harboring this rearrangement represented less than 0.6% of the whole TCR-alphabeta+ T cell repertoire. Kinetics analysis revealed that the expansion of lymphocytes bearing this conserved rearrangement was elicited specifically by donor blood transfusion. Indeed, Vbeta18-Dbeta1-Jbeta2.7 transcripts were detected in PBL from transfused animals as early as 7 days after donor-specific blood transfusion. Finally, we provided evidence that this T cell clone belongs to the CD8+ subset. The putative role in inducing and maintaining the allograft tolerance of the CD8+ T cell clone harboring this public Vbeta18-Dbeta1-Jbeta2.7 rearrangement is discussed.

Recombinant IFN-gamma abrogates allograft tolerance induced by donor-specific blood transfusion by restoring alloantibody production.

Donor-specific tolerance to heart allograft was induced in adult Lewis rats by pregraft donor-specific blood transfusion (DST). We previously showed that this tolerant state is characterized by a dramatic inhibition of T cell and macrophage activation. In addition, tolerant animals could not mount an efficient anti-donor humoral response whereas transfer of sera from rejecting animals triggered rejection in tolerant animals. This tolerance can be abrogated by daily post-graft administration of recombinant IFN-gamma (rIFN-gamma). To elucidate the mechanisms of action of rIFN-gamma, T cell, macrophage and B cell functions were assessed in allograft recipients. IFN-gamma did not restore the expression of Th1-related cytokine mRNA or the activated macrophage product inducible nitric oxide synthase in allografts. Importantly, rIFN-gamma treatment promptly restored the anti-donor humoral response in DST-treated recipients. We conclude that rIFN-gamma treatment in DST-treated allograft recipients cannot reverse the unresponsive state of Th1 cells and macrophages infiltrating the graft, but can provide B cell help for IgG alloantibody production which is lacking in these animals.

Anti-TCR Vbeta-specific DNA vaccination prolongs heart allograft survival in adult rats.

Allospecific T cells are known to play a central role in the process of allograft rejection. Recently, it has been shown that T cell function could be specifically targeted using DNA vaccination. In our model, PCR analysis of the TCR-beta chain repertoire of T cells infiltrating rejected allografts showed specific expansions of the Vbeta13 and Vbeta2 families. In this study, we tested the effect on allograft survival of DNA vaccination against a specific TCR Vbeta, in a model of heart allograft rejection in adult rats. Our results showed that anti-TCR Vbeta13 DNA vaccination lead to a significant prolongation of allograft survival compared to vaccination against other Vbeta families or untreated recipients. The prolongation of allograft survival correlated in vitro with a decrease in anti-donor reactivity of spleen cells from Vbeta13-vaccinated rats. These results show that, in a transplantation model, DNA vaccination could be used as a method to specifically manipulate a T cell response and thus prolong allograft survival.

T cell repertoire alterations of vascularized xenografts.

The role of T cells in the rejection of vascularized xenografts has been little explored. Because of the high potential diversity of xenoantigens, it has been suggested that xenotransplantation could induce a strong cellular response that could contribute to delayed rejection. Alternatively, alterations in molecular interactions could impair the T cell response. Because the analysis of TCR repertoire in vivo indirectly reflects the nature and the magnitude of T cell xenorecognition, we took advantage of the possibility of obtaining long term survival of hamster heart xenografts in rat recipients treated with a combination of cobra venom factor and cyclosporin A (CsA), to analyze T cell infiltration and, for the first time, V beta TCR usage, at the complementarity-determining region 3 level, in accommodated and rejected xenografts, compared with allografts. After withdrawal of CsA (on day 40), the analysis of V beta family expression and corresponding complementarity-determining region 3 lengths in rejected xenografts revealed a Gaussian pattern, in contrast to a much more restricted pattern in rejected allografts (p = 0.002), suggesting that, after withdrawal of CsA, all the underrepresented T cell clones are rapidly expanded in xenografts. These results correlate with the rapid kinetics of rejection (4 +/- 1 days), the high number of T cells, the rapid expression of markers of activation (IL-2 receptor alpha-chain and class II receptor), and the strong deposit of IgG Abs in rejected xenografts. Taken together, these results suggest that the intensity and diversity of the T cell response to xenografts could be stronger than the response to allografts in vivo.

The Cbl protooncogene product: from an enigmatic oncogene to center stage of signal transduction.

The c-cbl protooncogene was first identified as the cellular homologue of a viral oncogene v-cbl that induces pre-B lymphomas and myeloid leukemias in mice. Until recently, the biochemical basis for Cbl's transforming potential and its physiological role remained unclear. However, a convergence of biochemical studies in mammalian cells and genetic studies in C. elegans and Drosophila has now identified Cbl as a negative regulator of tyrosine kinase signaling. The N-terminal transforming region of Cbl (Cbl-N) and an adjacent RING finger domain are the elements most conserved during evolution. The Cbl-N region has now been shown to contain a novel phosphotyrosine-binding (PTB) domain that directly interacts with autophosphorylated tyrosine kinases via a D(N/D)XpY motif. A critical role of the PTB domain in Cbl function is demonstrated by the localization of a loss-of-function mutation in C. elegans Cbl homologue SLI-1 within this region. The corresponding mutation in human Cbl inactivates the PTB domain function and abrogates Cbl-mediated regulation of tyrosine kinase function. Recent studies have also identified a novel signaling pathway initiated by the interaction of mammalian Cbl proteins with the SH2 domains of Crk adaptor molecules, which results in Cbl's linkage with C3G, a guanine nucleotide exchange protein for Rap1 family of small G-proteins. Presently, Rap1 is thought to antagonize Ras function, although Rap1-specific targets have emerged recently. Thus, recent advances have firmly placed the little known protooncoprotein Cbl on the center stage of tyrosine kinase-mediated signal transduction.

Anti-TCR-specific DNA vaccination demonstrates a role for a CD8+ T cell clone in the induction of allograft tolerance by donor-specific blood transfusion.

Donor-specific allograft tolerance can be induced in the adult rat by pregraft donor-specific blood transfusion (DST). This tolerance appeared to be mediated by regulatory cells and to the production of the suppressive cytokine TGF-beta1. A potential immunoregulatory CD8+ clone bearing a Vbeta18-Dbeta1-Jbeta2.7 TCR gene rearrangement was previously identified in DST-treated recipients. To assess the functional role of this T cell clone in the induction of tolerance by DST, we have vaccinated DST-treated recipients with a plasmid construct encoding for the Vbeta18-Dbeta1-Jbeta2.7 TCR beta-chain. DST-induced allograft tolerance was abolished by anti-TCR Vbeta18-Dbeta1-Jbeta2.7 DNA vaccination in six of seven recipients, whereas vaccination with the vector alone, or with the construct encoding a TCR Vbeta13 beta-chain, had no effect. However, the transcript number of the Vbeta18-Dbeta1-Jbeta2.7 chain was unchanged in allografts from vaccinated DST-treated rats, suggesting that this clone was not depleted by vaccination, but rather was altered in its function. Moreover, TCR Vbeta18-Dbeta1-Jbeta2.7 DNA vaccination restored the anti-donor alloantibody production, partially restore the capacity of spleen cells from tolerized recipients to proliferate in vitro against donor cells, and decreased the inhibitory effect of TGF-beta1, seen in DST-treated recipients, in spleen cells from vaccinated DST-treated ones. This study strongly suggests that this CD8+ TCR Vbeta18-Dbeta1-Jbeta2.7 T cell clone has an effective immunoregulatory function in allograft tolerance induced by DST.