Autologous dendritic cells prolong allograft survival through Tmem176b-dependent antigen cross-presentation.

The administration of autologous (recipient-derived) tolerogenic dendritic cells (ATDCs) is under clinical evaluation. However, the molecular mechanisms by which these cells prolong graft survival in a donor-specific manner is unknown. Here, we tested mouse ATDCs for their therapeutic potential in a skin transplantation model. ATDC injection in combination with anti-CD3 treatment induced the accumulation of CD8(+) CD11c(+) T cells and significantly prolonged allograft survival. TMEM176B is an intracellular protein expressed in ATDCs and initially identified in allograft tolerance. We show that Tmem176b(-/-) ATDCs completely failed to trigger both phenomena but recovered their effect when loaded with donor peptides before injection. These results strongly suggested that ATDCs require TMEM176B to cross-present antigens in a tolerogenic fashion. In agreement with this, Tmem176b(-/-) ATDCs specifically failed to cross-present male antigens or ovalbumin to CD8(+) T cells. Finally, we observed that a Tmem176b-dependent cation current controls phagosomal pH, a critical parameter in cross-presentation. Thus, ATDCs require TMEM176B to cross-present donor antigens to induce donor-specific CD8(+) CD11c(+) T cells with regulatory properties and prolong graft survival.

Cell therapy with autologous tolerogenic dendritic cells induces allograft tolerance through interferon-gamma and epstein-barr virus-induced gene 3.

Innovative therapeutic strategies are needed to diminish the impact of harmful immunosuppression in transplantation. Dendritic cell (DC)-based therapy is a promising approach for induction of antigen-specific tolerance. Using a heart allograft model in rats, we analyzed the immunoregulatory mechanisms by which injection of autologous tolerogenic DCs (ATDCs) plus suboptimal immunosuppression promotes indefinite graft survival. Surprisingly, we determined that Interferon-gamma (IFNG), a cytokine expected to be propathogenic, was threefold increased in the spleen of tolerant rats. Importantly, its blockade led to allograft rejection [Mean Survival Time (MST) = 25.6 ± 4 days], showing that IFNG plays a critical role in immunoregulatory mechanisms triggered by ATDCs. IFNG was expressed by TCRαß(+) CD3(+) CD4(-) CD8(-) NKRP1(-) cells (double negative T cells, DNT), which accumulated in the spleen of tolerant rats. Interestingly, ATDCs specifically induced IFNG production by DNT cells. ATDCs expressed the cytokinic chain Epstein-Barr virus-induced gene 3 (EBI3), an IL-12 family member. EBI3 blockade or knock-down through siRNA completely abolished IFNG expression in DNT cells. Finally, EBI3 blockade in vivo led to allograft rejection (MST = 36.8 ± 19.7 days), demonstrating for the first time a role for EBI3 in transplantation tolerance. Taken together our results have important implications in the rationalization of DC-based therapy in transplantation as well as in the patient immunomonitoring follow-up.

Long-term allograft tolerance is characterized by the accumulation of B cells exhibiting an inhibited profile.

Numerous reports have highlighted the central role of regulatory T cells in long-term allograft tolerance, but few studies have investigated the B-cell aspect. We analyzed the B-cell response in a rat model of long-term cardiac allograft tolerance induced by a short-term immunosuppression. We observed that tolerated allografts are infiltrated by numerous B cells organized in germinal centers that are strongly regulated in their IgG alloantibody response. Moreover, alloantibodies from tolerant recipients exhibit a deviation toward a Th2 isotype and do not activate in vitro donor-type endothelial cells in a pro-inflammatory way but maintained expression of cytoprotective molecules. Interestingly, this inhibition of the B-cell response is characterized by the progressive accumulation in the graft and in the blood of B cells blocked at the IgM to IgG switch recombination process and overexpressing BANK-1 and the inhibitory receptor Fcgr2b. Importantly, B cells from tolerant recipients are able to transfer allograft tolerance. Taken together, these results demonstrate a strong regulation of the alloantibody response in tolerant recipients and the accumulation of B cells exhibiting an inhibited and regulatory profile. These mechanisms of regulation of the B-cell response could be instrumental to develop new strategies to promote tolerance.

Interaction of Fc receptor (CD16) ligands induces transcription of interleukin 2 receptor (CD25) and lymphokine genes and expression of their products in human natural killer cells.

We report evidence that FcR(CD16) on human NK cells are signal-transducing molecules that, upon ligand binding, induce transcription of genes encoding surface activation molecules [IL-2-R(CD25)] and cytokines (IFN-gamma and TNF) relevant to NK cell biology and functions. Homogeneous NK and T cell populations purified from short-term bulk cultures of PBMC with irradiated B lymphoblastoid cell lines were cultured in the presence of FcR ligands (particulate immune complexes or immobilized anti-CD16 antibodies) alone or with rIL-2. Upon 18 h of stimulation, NK cells express Tac, TfR, and 4F2 antigens and produce IFN-gamma and TNF; both effects are synergistically enhanced in the presence of rIL-2, which is itself ineffective. Treatment of NK cells with FcR(CD16) ligands induces accumulation of mRNA for IFN-gamma and TNF and, to a lesser extent, IL-2-R with fast kinetics also in the absence of de novo protein synthesis. rIL-2 and FcR(CD16) ligands synergize to induce mRNA accumulation. mRNA accumulation and transcription of TNF and IFN-gamma genes induced by FcR(CD16) ligands are greater than those induced by rIL-2, and the reverse is true for IL-2-R. The two stimuli do not synergize at the transcriptional level. These observations indicate that the mechanisms through which FcR(CD16) ligands and rIL-2 induce NK cell activation are, in part, distinct. Both operate at the transcriptional level, although other mechanisms are probably induced by the FcR ligand stimulus per se or in combination with other lymphokines and synergize at a post-transcriptional or translational level to enhance NK cell activation.

Rat spleen dendritic cells express natural killer cell receptor protein 1 (NKR-P1) and have cytotoxic activity to select targets via a Ca2+-dependent mechanism.

Dendritic cells (DC) are a subset of leukocytes whose major function is antigen presentation. We investigated the phenotype and function of enriched (95-98.5%) rat DC. We show that both spleen and thymus DC express the natural killer cell receptor protein 1 (NKR-P1) as a disulfide linked homodimer of 60 kD. Freshly isolated DC express a low level of NKR-P1, which is strongly upregulated after overnight culture. Spleen, but not thymus DC, were able to kill the NK-sensitive YAC-1 cell line in vitro, and since this killing was Ca2+ dependent, a Fas ligand-Fas interaction was probably not involved. Besides their potent antigen-presenting function, DC can thus be cytotoxic for some tumor targets.

Independent regulation of tumor necrosis factor and lymphotoxin production by human peripheral blood lymphocytes.

We present evidence that human peripheral blood lymphocytes, free of contaminating monocytes, rapidly produce high levels of tumor necrosis factor (TNF) when stimulated with phorbol diester and calcium ionophore, and lower but significant levels of TNF when stimulated with mitogens. These two types of inducers act preferentially on T cells, both CD4+ and CD8+. NK cells produce TNF only when stimulated with phorbol diester and calcium ionophore, and they do so at a much lower level than T cells. The procedures used in the purification of lymphocytes and the differential ability to respond to various inducers allow us to exclude that monocytes or basophils contaminating the lymphocyte preparation participate in the production of TNF. In particular, LPS, a potent inducer of TNF production from monocytes, is unable to induce significant levels of TNF in the lymphocyte preparations. The TNF produced by lymphocytes has antigenic, physicochemical, and biochemical characteristics identical to those of the TNF produced by myeloid cell lines or monocytes upon stimulation with LPS. LT is also produced by lymphocyte preparations. Production of TNF and LT proteins in response to the different inducers is paralleled by accumulation of cytoplasmic TNF and LT mRNA. Both at mRNA and at protein levels, stimulation of T lymphocytes with phorbol diester and calcium ionophore preferentially induces TNF, whereas mitogen stimulation preferentially induces LT. Our data suggest that the TNF and LT genes, two closely linked genes encoding two partially homologous proteins with almost identical biological functions, are independently regulated in lymphocytes.

Fc gamma R(CD16) interaction with ligand induces Ca2+ mobilization and phosphoinositide turnover in human natural killer cells. Role of Ca2+ in Fc gamma R(CD16)-induced transcription and expression of lymphokine genes.

In this study, we present evidence that interaction of Fc gamma R(CD16) with ligands (immune complexes or anti-CD16 antibodies) induces a rapid rise in [Ca2+]i and fast production of both inositol 1,4,5 triphosphate (IP3) and IP4 in homogeneous NK cell preparations. Part of the initial [Ca2+]i rise observed upon stimulation of NK cells with either anti-CD16 antibodies alone or after their crosslinking at the cell membrane depends on Ca2+ mobilization from intracellular stores, but sustained [Ca2+]i levels are maintained, after the initial spike, through influx of extracellular Ca2+. The [Ca2+]i rise is mediated, at least in part, by increases in IP3 after receptor-induced hydrolysis of membrane polyphosphoinositides (PPI). The role of extracellular Ca2+ in Fc gamma R(CD16)-dependent induction of lymphokine gene expression has been tested by evaluating production, mRNA accumulation and transcription of IFN-gamma and TNF in NK cells stimulated with Fc gamma R(CD16) ligands and/or rIL-2 in the presence of EGTA. Under these conditions, accumulation and transcription of both IFN-gamma and TNF mRNA induced by CD16 ligands, but not that induced by rIL-2, is completely abolished and neither cytokine can be detected at significant levels in the supernatant fluids of cells so treated. These data confirm that NK cell activation by specific ligands occurs through mechanisms distinct from those induced by IL-2, and indicate that extracellular Ca2+ represents a stringent requirement for cytokine production induced in NK cells through specific (Fc gamma R) stimulation. Our data also indicate that the [Ca2+]i rise induced upon Fc gamma R(CD16) crosslinking, though necessary, is not sufficient per se to induce activation of lymphokine genes, compatible with the hypothesis that Fc gamma R(CD16) crosslinking generates additional transducing signals that synergize with IL-2 to maximally activate NK cells.

Production of hematopoietic colony-stimulating factors by human natural killer cells.

We have analyzed the ability of highly purified preparations of human NK cells to produce CSF. NK cells, purified by negative selection from 10-d cultures of PBMC incubated with irradiated B-lymphoblastoid cell lines, were stimulated with rIL-2, FcR(CD16) ligands (particulate immune complexes or anti-CD16 antibodies bound to Sepharose), a combination of CD16 ligands and rIL-2, or the phorbol diester phorbol dibutyrate (PDBu) together with the Ca2+ ionophore A23187. Both rIL-2 and CD16 ligands induce accumulation of GM-CSF mRNA in NK cells and the combined effect of the two stimuli is synergistic. Maximal accumulation of GM-CSF mRNA is observed after PDBu/A23187 stimulation. The participation of contaminant T cells in the observed expression of the GM-CSF gene is excluded because CD16 ligands do not stimulate T cells and CD3 ligands, powerful stimulators of T cells, are inactive on NK cells. Accumulation of CSF-1 mRNA is observed only in NK cells stimulated with both CD16 ligands and rIL-2, whereas accumulation of IL-3 mRNA is observed only in NK cells stimulated with PDBu/A23187. Transcripts of the G-CSF, IL-1 alpha, and IL-1 beta genes were never detected in NK cells in these experiments. The kinetics of accumulation of GM-CSF and CSF-1 mRNA in NK cells stimulated with CD16 ligands and rIL-2 peaked at 2-4 h and was slower than that of TNF and IFN-gamma mRNA, which peak at 1 h. GM-CSF was precipitated from the supernatant fluids of NK cells stimulated with PDBu/A23187 and its biological activity was demonstrated by the ability of the supernatants to sustain proliferation of the TALL-101 cell line or CML blasts. Biological activity of IL-3 and CSF-1 was demonstrable in supernatant fluids of NK cells stimulated with PDBu/A23187 and CD16 ligands/rIL-2, respectively.

Tolerogenic dendritic cells actively inhibit T cells through heme oxygenase-1 in rodents and in nonhuman primates.

Clinical translation of dendritic cell (DC)-based cell therapy requires preclinical studies in nonhuman primates (NHPs). The aim of this work was to establish the in vitro conditions for generation of NHP tolerogenic DCs (Tol-DCs), as well as to analyze the molecular mechanisms by which these cells could control an immune response. Two populations of NHP bone marrow-derived DCs (BMDCs) were obtained: adherent and nonadherent. Although both populations displayed a quite similar phenotype, they were very different functionally. We characterized the adherent BMDCs as Tol-DCs that were poor stimulators of T cells and actively inhibited T-cell proliferation, whereas the nonadherent population displayed immunogenic properties in vitro. Interestingly, the anti-inflammatory and immunosuppressive enzyme heme oxygenase-1 (HO-1) was up-regulated in Tol-DCs, compared to the immunogenic BMDCs. We demonstrated that HO-1 mediates the immunosuppressive properties of Tol-DCs in vitro (in NHPs and rats) and that HO-1 is involved in the in vivo tolerogenic effect of Tol-DCs in a rat model of allotransplantation. In conclusion, here we characterized the in vitro generation of NHP Tol-DCs. Furthermore, we showed for the first time that HO-1 plays a role in the active inhibition of T-cell responses by rat and NHP Tol-DCs.

Endotoxin-induced myeloid-derived suppressor cells inhibit alloimmune responses via heme oxygenase-1.

Inflammation and cancer are associated with impairment of T-cell responses by a heterogeneous population of myeloid-derived suppressor cells (MDSCs) coexpressing CD11b and GR-1 antigens. MDSCs have been recently implicated in costimulation blockade-induced transplantation tolerance in rats, which was under the control of inducible NO synthase (iNOS). Herein, we describe CD11b+GR-1+MDSC-compatible cells appearing after repetitive injections of lipopolysaccharide (LPS) using a unique mechanism of suppression. These cells suppressed T-cell proliferation and Th1 and Th2 cytokine production in both mixed lymphocyte reaction and polyclonal stimulation assays. Transfer of CD11b+ cells from LPS-treated mice in untreated recipients significantly prolonged skin allograft survival. They produced large amounts of IL-10 and expressed heme oxygenase-1 (HO-1), a stress-responsive enzyme endowed with immunoregulatory and cytoprotective properties not previously associated with MDSC activity. HO-1 inhibition by the specific inhibitor, SnPP, completely abolished T-cell suppression and IL-10 production. In contrast, neither iNOS nor arginase 1 inhibition did affect suppression. Importantly, HO-1 inhibition before CD11b+ cell transfer prevented the delay of allograft rejection revealing a new MDSC-associated suppressor mechanism relevant for transplantation.

Impaired Notch4 activity elicits endothelial cell activation and apoptosis: implication for transplant arteriosclerosis.

OBJECTIVE: Notch signaling pathway controls key functions in vascular and endothelial cells (EC). However, little is known about the role of Notch in allografted vessels during the development of transplant arteriosclerosis (TA). This study investigated regulation of the Notch pathway on cardiac allograft arteriosclerosis and further examined its implication in EC dysfunction. METHODS AND RESULTS: Here we show that, among Notch receptors, Notch2, -3, and -4 transcript levels were markedly downregulated in TA compared to tolerant and syngeneic allografts. TA correlates with high levels of tumor necrosis factor (TNF), transforming growth factor (TGF)beta, and IL10, which consistently decrease Notch4 expression in transplants and cultured ECs. We found that inhibition of Notch activity, reflected by both a reduced CBF1 activity and Hes1 expression, parallels the downregulation of Notch4 expression mediated by TNF in ECs. Notch4 and Hes1 knockdown enhances vascular cell adhesion molecule-1 expression and promotes EC apoptosis. Silencing Notch4 or Hes1 also drastically inhibits repair of endothelial injury. Overall, our results suggest that Notch4 and basal Notch activity are required to maintain EC quiescence and for optimal survival and repair in response to injury. CONCLUSIONS: Together, our findings indicate that impaired Notch4 activity in graft ECs is a key event associated with TA by triggering EC activation and apoptosis.

An immunomodulatory role for follistatin-like 1 in heart allograft transplantation.

Donor-specific tolerance to heart allografts in the rat can be achieved by donor-specific blood transfusions (DST) before transplantation. We have previously reported that this tolerance is associated with strong leukocyte infiltration, and that host CD8(+) T cells and TGFbeta are required. In order to identify new molecules involved in the induction phase of tolerance, we compared tolerated and rejected heart allografts (suppressive subtractive hybridization) 5 days after transplantation. We identified overexpression of Follistatin-like 1 (FSTL1) transcript in tolerated allografts compared to rejected allografts or syngeneic grafts. We show that FSTL1 is overexpressed during both the induction and maintenance phase of tolerance, and appears to be specific to the tolerance model induced by DST. Analysis of graft-infiltrating cells revealed predominant expression of FSTL1 in CD8(+) T cells from tolerated grafts, and depletion of these cells prior to transplantation abrogated FSTL1 expression and heart allograft survival. Moreover, overexpression of FSTL1 by adenovirus gene transfer in vivo significantly prolonged allograft survival in association with inhibition of the proinflammatory cytokines, IL6, IL17 A and IFNgamma. Taken together, these results suggest that FSTL1 could be an active component of the mechanisms mediating heart allograft tolerance.

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.

Immune Cells and Molecular Networks in Experimentally Induced Pulpitis.

Dental pulp is a dynamic tissue able to resist external irritation during tooth decay by using immunocompetent cells involved in innate and adaptive responses. To better understand the immune response of pulp toward gram-negative bacteria, we analyzed biological mediators and immunocompetent cells in rat incisor pulp experimentally inflamed by either lipopolysaccharide (LPS) or saline solution (phosphate-buffered saline [PBS]). Untreated teeth were used as control. Expression of pro- and anti-inflammatory cytokines, chemokine ligands, growth factors, and enzymes were evaluated at the transcript level, and the recruitment of the different leukocytes in pulp was measured by fluorescence-activated cell-sorting analysis after 3 h, 9 h, and 3 d post-PBS or post-LPS treatment. After 3 d, injured rat incisors showed pulp wound healing and production of reparative dentin in both LPS and PBS conditions, testifying to the reversible pulpitis status of this model. IL6, IL1-ß, TNF-α, CCL2, CXCL1, CXCL2, MMP9, and iNOS gene expression were significantly upregulated after 3 h of LPS stimulation as compared with PBS. The immunoregulatory cytokine IL10 was also upregulated after 3 h, suggesting that LPS stimulates not only inflammation but also immunoregulation. Fluorescence-activated cell-sorting analysis revealed a significant, rapid, and transient increase in leukocyte levels 9 h after PBS and LPS stimulation. The quantity of dendritic cells was significantly upregulated with LPS versus PBS. Interestingly, we identified a myeloid-derived suppressor cell-enriched cell population in noninjured rodent incisor dental pulp. The percentage of this population, known to regulate immune response, was higher 9 h after inflammation triggered with PBS and LPS as compared with the control. Taken together, these data offer a better understanding of the mechanisms involved in the regulation of dental pulp immunity that may be elicited by gram-negative bacteria.

Characterization of a human monocyte antigen, B148.4, regulated during cell differentiation and activation.

We analyzed the phenotypic changes associated with monocyte activation and differentiation using a newly developed monoclonal antibody (B148.4). Among peripheral blood leukocytes, the antigen recognized by this antibody is expressed on monocytes and granulocytes at high and low density, respectively. Antigen expression is lost during in vitro differentiation of monocytes and is absent on tissue macrophages, indicating that expression of this antigen is related to monocyte differentiation. Only 1 alpha,25-dihydroxyvitamin D3 and phorbol diesters, of several inducers tested, up-regulate B148.4 antigen expression on cells (monocytes and certain myeloid cell lines) that constitutively bear the antigen, without, however, allowing its maintenance during monocytic differentiation or inducing it on negative cells. By immunoprecipitation from B148.4+ U937 cells, the antigen is a complex of a major 116-kd and two minor 38- and 46-kd molecules. Analysis of eight different tissues reveals that the antigen is shared with endothelial cells. Biochemical characteristics, cellular distribution, and expression pattern on monocytes, myeloid cell lines, and AML cells upon culture with different stimuli indicate that B148.4 is a novel monocyte differentiation antigen.

Similar levels of granzyme A and perforin mRNA expression in rejected and tolerated heart allografts in donor-specific tolerance in rats.

Congenic LEW.1W (RT1u) heart grafts in LEW.1A (RT1u) recipient rats are rejected within 15 +/- 6 days. Tolerance (> 100 days) can be induced by pretransplant donor-specific blood transfusions. In both cases, the graft is heavily infiltrated by recipient cells, and class I and class II molecules of the major histocompatibility complex are strongly expressed. Moreover, T lymphocytes extracted from both tolerated and rejected grafts are similarly cytotoxic in vitro against donor cells. However, it cannot be excluded that this cytotoxicity does not operate in vivo. To answer this important question, we have studied the expression of granzyme A and perforin mRNA expression, in situ, by Northern blotting. Our data show that the two corresponding mRNAs accumulate with the same kinetic and at the same level in rejected and tolerated grafts. These results strongly suggest that infiltrating cells are cytotoxic in vivo and that a "cellular rejection" does occur in the "tolerated" grafts. In addition, these findings show that cytotoxic T lymphocytes are not sufficient to impair graft survival and lead to a revaluation of the relevance, in general, terms of granzyme and perforin expression as a correlate of rejection.

Long-term survival of hamster-to-rat cardiac xenografts in the absence of a Th2 shift.

BACKGROUND: In hamster-to-rat cardiac xenografts, long-term survival (LTS) is obtained in 60% of recipients if vascular rejection is overcome by cobra venom factor and cyclosporine (CsA). It has been suggested that this accommodation state could be due to the Th2 response. METHODS: We examined the infiltrate by using immunostaining and the accumulation of cytokine mRNA (interferon-gamma [IFN-gamma], interleukin [IL]-4, IL-10, IL-13, and transforming growth factor-betal [TGF-beta1]) by using competitive reverse transcriptase polymerase chain reaction, in hamster hearts grafted into LEW.1A rat. RESULTS: Hearts from untreated and treated (cobra venom factor and CsA) but rejecting recipients presented a rapid and severe vascular rejection. In contrast, hearts from long-surviving treated animals had subnormal cardiac muscle with a mild infiltrate, principally macrophages, which peaked on day 15. T lymphocytes were also maximal on day 15 (12% of the infiltrate). Rejected grafts from untreated recipients showed accumulation of IFN-gamma mRNA but low levels of IL-10, TGF-beta, and IL-13. In hearts rejected by treated recipients, IFN-gamma mRNA did not increase and TGF-beta mRNA was higher. In LTS, IL-10, TGF-beta, and IL-13 transcripts were up-regulated (P<0.001), while IFN-gamma mRNA decreased (P<0.001). In both groups, IL-4 expression remained at a nonsignificant level. CONCLUSIONS: The profile of cytokine mRNAs in LTS could result in part from CsA, known to up-regulate TGF-beta and to down-regulate IFN-gamma. Moreover, CsA does not inhibit IL-10 production by monocyte/macrophages, the major infiltrating cells (60%). Lastly, LTS is induced in the absence of IL-4, which suggests that the high IL-4 production could simply be correlated with LTS without being a condition for it.

Prolongation of heart xenograft survival in a hamster-to-rat model after therapy with a rationally designed immunosuppressive peptide.

BACKGROUND: Modification of the aminoacid sequence of peptides derived from the HLA class I heavy chain in combination with computer rational design resulted in the development of a peptide, RDP1258, with enhanced immunosuppressive activity. METHODS: We evaluated the activity of this peptide, analyzing infiltrate by immunohistology and cytokine transcripts by reverse transcriptase-polymerase chain reaction method, in a hamster-to-rat xenograft model where recipients were treated with cobra venom factor (CVF) and peptide. RESULTS: Although CVF or peptide alone had no effect, a combination of CVF/peptide RDP1258 resulted in a significant prolongation of graft survival (7.9+/-1 vs. 4.5+/-0 and 3.5+/-0 days, P<0.001). This effect was associated with an increased expression of heme oxygenase 1 (HO-1) in spleen, a significant reduced graft infiltrate, and a decrease of tumor necrosis factor-alpha mRNA transcripts (P<0.05) compared with CVF-treated recipients (1.6+/-0.07 vs. 3.3+/-0.3%, P=0.001) on day 3 after transplantation. CONCLUSION: These observations are consistent with the observation that up-regulation of HO-1 results in inhibition of immune effector functions and suggest that the peptide acts, at least partially, through HO-1 regulation.

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.

Peripheral tolerance of an allograft in adult rats--characterization by low interleukin-2 and interferon-gamma mRNA levels and by strong accumulation of major histocompatibility complex transcripts in the graft.

Congenic LEW.1W(RT1.u) heart grafts in LEW.1A(RT1.a) recipient rats are rejected in 15 +/- 6 days. Tolerance (greater than 100 days) can be induced by pretransplant donor-specific blood transfusion. In this case, the graft is not rejected, although it is infiltrated by mononuclear cells specifically cytotoxic, in vitro, against allogeneic donor splenocytes. We studied the expression of MHC class I and class II antigens, IFN-gamma, and IL-2 mRNA in the rejected and tolerated grafts by Northern blotting and in situ hybridization. Our data show that both class I and class II mRNA accumulate in both types of graft, and that class I mRNA accumulation occurs more rapidly in the tolerated grafts. IFN-gamma and IL-2 mRNA accumulate to lower levels and with delayed kinetics in the tolerated grafts compared with the rejected ones, suggesting a role for these lymphokines in the mechanism of rejection/tolerance in this model. This hypothesis is also supported by the observation that IFN-gamma treatment abrogates the induction of tolerance in the recipients receiving pretransplant donor blood transfusion. Furthermore, we observed an uncoupling of the accumulation of IFN-gamma mRNA and of MHC class I and class II mRNA. Our data confirm that the mechanisms of tolerance in this model depend, in part, on alterations of the IL-2/IL-2R pathway of lymphocyte activation but also clearly indicate a decrease of IFN-gamma mRNA accumulation, suggesting that the defect involves several activation molecules.