Regulatory functions of self-restricted MHC class II allopeptide-specific Th2 clones in vivo.

We studied T-cell clones generated from grafts of rejecting and tolerant animals and investigated the regulatory function of Th2 clones in vitro and in vivo. To prevent allograft rejection, we treated LEW strain recipient rats of WF strain kidney grafts with CTLA4Ig to block CD28-B7 costimulation. We then isolated epitope-specific T-cell clones from the engrafted tissue, using a donor-derived immunodominant class II MHC allopeptide presented by recipient antigen-presenting cells. Acutely rejected tissue from untreated animals yielded self-restricted, allopeptide-specific T-cell clones that produced IFN-gamma, whereas clones from tolerant animals produced IL-4 and IL-10. Adoptive transfer into naive recipients of Th1 clones, but not Th2 clones, induced alloantigen-specific delayed-type hypersensitivity (DTH) responses. In addition, Th2 clones suppressed DTH responses mediated by Th1 clones in vivo and blocked Th1 cell proliferation and IFN-gamma production in vitro. A pilot human study showed that HLA-DR allopeptide-specific T-cell clones generated from patients with chronic rejection secrete Th1 cytokines, whereas those from patients with stable graft function produce Th2 cytokines in response to donor-specific HLA-DR allopeptides. We suggest that self-restricted alloantigen-specific Th2 clones may regulate the alloimmune responses and promote long-term allograft survival and tolerance.

CD28-B7-mediated T cell costimulation in chronic cardiac allograft rejection: differential role of B7-1 in initiation versus progression of graft arteriosclerosis.

Provision of adequate T cell costimulation is critical for the development of acute and chronic allograft rejection. We have previously reported that early blockade of CD28-B7 T cell costimulation prevents the development of graft arteriosclerosis, in the LEW into F344 rat cardiac transplant model. In this study, we used the same model to examine the requirement for CD28-B7-mediated T cell costimulation in the progression of established chronic rejection and examined the individual roles of B7-1 (CD80) and B7-2 (CD86) costimulatory molecules. Late blockade of CD28-B7 T cell costimulation by the fusion protein CTLA4Ig, which binds both CD80 and CD86, attenuated the development of transplant arteriosclerosis, mononuclear cell infiltration, and parenchymal fibrosis in this model. Selective blockade of CD80 using the mutant fusion protein Y100F was as effective as CTLA4Ig in this regard. In contrast to CTLA4Ig, blockade of CD80 alone by Y100F was ineffective at preventing early graft loss and prolonging graft survival when given early after transplantation. This study is the first to demonstrate that late blockade of CD28-B7 T cell costimulation interrupts chronic cardiac allograft rejection, and it indicates the importance of continued T cell activation in this process. This study further defines functional differences between CD80 and CD86 costimulatory molecules in vivo.

Mechanisms of chronic rejection.

Chronic rejection remains the major obstacle to long-term allograft survival. Detailed understanding of putative etiologic risk factors, both antigen-dependent and -independent, is important for designing effective therapeutic strategies to ameliorate this process. Cell senescence may be an important factor in chronic rejection.

The role of CD154-CD40 versus CD28-B7 costimulatory pathways in regulating allogeneic Th1 and Th2 responses in vivo.

We used signal transducer and activator of transcription 4 (STAT4) and STAT6 gene knockout (-/-) mice as recipients of fully mismatched cardiac allografts to study the role of T-cell costimulatory pathways in regulating allogeneic T-helper 1 (Th1) versus Th2 responses in vivo. STAT4(-/-) mice have impaired Th1 responses, whereas STAT6(-/-) mice do not generate normal Th2 responses. Cardiac allografts from C57BL/6 mice were transplanted into normal wild-type (WT), STAT4(-/-), and STAT6(-/-) BALB/c recipients. STAT4(-/-) and STAT6(-/-) mice rejected their grafts with the same tempo as untreated WT recipients. CD28-B7 blockade by a single injection of CTLA4Ig induced long-term engraftment and donor-specific tolerance in all three groups of recipients. CD154 blockade by a single injection of MR1 was effective in prolonging allograft survival and inducing tolerance in STAT4(-/-) mice but was only marginally effective in STAT6(-/-) recipients and WT controls. In addition, a similar protocol of MR1 was ineffective in prolonging graft survival in CD28(-/-) BALB/c recipients, suggesting that the lack of efficacy seen in WT and STAT6(-/-) mice is not due to the presence of a functional CD28-B7 pathway. Furthermore, there was a similar differential effect of CD28-B7 versus CD154-CD40 blockade in inhibiting immune responses in animals immunized with ovalbumin and complete Freund's adjuvant. These novel data indicate that Th1 and Th2 cells are differentially regulated by CD28-B7 versus CD154-CD40 costimulation pathways in vivo and may have potential implications for the development of therapeutic strategies such as T-cell costimulatory blockade in humans.

The influence of donor brain death on short and long-term outcome of solid organ allografts.

Long-term survival rates of solid organ allografts have improved relatively little during the transplant experience despite more effective immunosuppression, better organ preservation techniques and advances in perioperative management. Because grafts of potentially diminished quality are increasingly accepted to reduce the severe shortage of organs, it has become apparent that a variety of donor-associated risk factors may influence adversely their short and long-term outcome. Recent interest has focused particularly on systemic changes occurring after donor brain death (BD). Numbers of experimental and clinical studies have elucidated the complexities of the hemodynamic, metabolic, neurohormonal, and other physiological alterations following this devastating central injury. This article will address the potential derangements in peripheral organs which may influence their behavior after transplantation.

Inhibition of allorecognition by a human class II MHC-derived peptide through the induction of apoptosis.

The interaction of the T-cell receptor with the major histocomatibility complex (MHC)-peptide complex is central to T-cell activation. Variation in the nature of the peptide bound within the groove of the MHC molecule may result in an altered T-cell response. Because some naturally processed peptides bound within the groove of the class II MHC molecule are derived from the MHC molecules themselves, we studied the inhibitory effects of synthetic class II MHC peptides on alloimmune responses in vitro. Three peptides derived from a highly conserved region of the class II MHC alpha chains inhibited the rat mixed lymphocyte response (MLR) in a dose-dependent manner, with the human HLA-DQA1 peptide also inhibiting the human and mouse MLR. No effect was seen on mitogen-induced T-cell proliferation. HLA-DQA1 inhibited cytolytic T lymphocyte (CTL) generation in a dose-response fashion, with no reduction in preformed CTL killing, suggesting that the inhibitory effect is targeted at CD4(+) T-cell function. Cell-cycle analysis by flow cytometry showed that restimulation of primed T cells in the presence of HLA-DQA1 resulted in increased apoptosis, whereas unstimulated cells were not affected. These data demonstrate that synthetic peptides derived from highly conserved regions of the class II MHC alpha chain can alter CD4(+) T-lymphocyte alloimmune responses in vitro, and this effect is mediated by the induction of apoptosis in activated T cells.

Comparative studies of specific acquired systemic tolerance induced by intrathymic inoculation of a single synthetic Wistar-Furth (RT1U) allo-MHC class I (RT1.AU) peptide or WAG (RT1U)-derived class I peptide.

BACKGROUND: Because T cell receptor-MHC class I/self-peptide interactions regulate T-cell development, the presence of MHC allopeptides in the thymus may influence T-cell tolerance to alloantigens. This hypothesis is supported by our most recent finding that intrathymic (IT) inoculation of nonimmunogenic synthetic peptides derived from "WAG" RT1.A induces tolerance to cardiac allografts in the Wistar-Furth (WF)-to-ACI model. To evaluate whether in vivo immunogenicity of MHC peptides is relevant to tolerance induction and to examine the effect of peptide specificity, we compared the effects on graft survival of well-defined, strain-specific immunogenic WF MHC class I peptides (RT1.AU) with closely related but non-strain-specific class I peptides derived from WAG (RT1U). METHODS: In vivo immunization of seven MHC class I peptides synthesized from RT1.AU sequences showed that two (u-5 and u-7) were immunogenic, whereas five others were not immunogenic in ACI recipients. We then examined the effects on cardiac allograft survival in the WF-to-ACI model of the two immunogenic RT1.AU peptides (u-5 and u-7) and three immunogenic WAG-derived peptides (peptides 1, 2, and 5). RESULTS: A combination of equal amounts (150 microg or 300 microg) of u-5 or u-7 each with 0.5 ml of antilymphocyte serum (ALS) on day -7 led to 60% and 100% permanent graft survival (>150 days), respectively. IT injection of the individual peptides on day -7 showed that only 300 microg of u-5 significantly prolonged graft survival to a median survival time of 17.3 days from 10.5 days in naive recipients. IT injection of 150, 300, and 600 microg of u-5 combined with 0.5 ml of ALS on day -7 led to permanent graft survival (> 150 days) in four of six, nine of nine, and six of six ACI recipients, respectively, compared with a median survival time of 15.4 days in ALS alone-treated controls. In contrast, similar treatments with peptide u-7 with or without 0.5 ml of ALS did not prolong graft survival, thus demonstrating that peptide u-5 alone mediates the observed effects on graft prolongation. A total of 300 microg of u-5 injected IT combined with ALS led to acute rejection of third-party (Lewis) grafts. Intravenous injection of 300 microg of u-5 with ALS also did not prolong WF graft survival in ACI recipients. The long-term unresponsive ACI recipients accepted permanently donor-type (WF) but not third-party (Lewis) second-set cardiac and islet allografts. Similarly, we showed that although IT injection of 600 and 1200 microg of a mixture of immunogenic WAG-derived peptides 1, 2, and 5 combined with 0.5 ml of ALS on day -7 led to permanent WF graft survival in ACI, only IT injection of 300 microg of peptide 2 combined with ALS led to permanent graft survival (>150 days) in four of five animals. To define the underlying mechanisms of tolerance, we examined in vitro the mixed lymphocyte reaction (MLR), cell-mediated lymphocytotoxicity, and cytokine profile of unresponsive recipients. Although the results showed nonspecific T-cell suppression in the MLR at 25 days after transplantation, which correlated with the persistence of ALS immunosuppression, long-term unresponsive animals showed normal MLR to donor and third-party antigens. In contrast, the donor-specific reactive cytotoxic T lymphocytes remained suppressed in short-term and long-term unresponsive rats. CONCLUSION: Of interest is our finding that IT injection of a short segment of WAG-derived MHC class I peptide induces active acquired tolerance similar to results obtained with the use of pure WF-derived peptide u-5 in the WF-to-ACI rat combination. It is noteworthy that we could not confirm the T helper (Th)1/Th2 paradigm in this model by initial cytokine analysis. Whether induction of tolerance by IT injection of allo-MHC peptides will have clinical usefulness must await results of similar studies in large animals. However, of major interest is the finding that a short segment of RT1.AU represents the tolerogenic

Effects of explosive brain death on cytokine activation of peripheral organs in the rat.

BACKGROUND: The success rate of transplanted organs from brain-dead cadaver donors is consistently inferior to that of living sources. As cadaver and living unrelated donors are equally genetically disparate with a given recipient, the difference must lie within the donor himself and/or the effects of organ preservation and storage. We have hypothesized that irreversible central nervous system injury may up-regulate proinflammatory mediators and cell surface molecules in peripheral organs to be engrafted, making them more prone to host inflammatory and immunological responses. METHODS: Rats undergoing surgically induced acutely increased intracranial pressure (explosive brain death) were followed for 6 hr. Their peripheral tissues were examined by reverse transcriptase polymerase chain reaction and immunohistology, serum factors were assessed by enzyme-linked immunosorbent assay, and the influence of inflammatory molecules in the blood stream was determined by cross-circulation experiments with normal animals. RESULTS: mRNA expression of both lymphocyte- and macrophage-associated products increased dramatically in all tissues. Similar factors in serum were coincidentally increased; these were shown to be active in vivo by cross-circulation with normal animals. The organs of all control groups, including animals with important ischemic injury and with hemorrhagic shock, were negative. Up-regulation of MHC class I and II antigens and the co-stimulatory molecule B7 suggests increased immunogenicity of the peripheral organs. These changes could be inhibited by: (i) administration of a recombinant soluble P-selectin glycoprotein ligand-Ig, a P- and E-selectin antagonist; and (ii) a fusion protein, cytotoxic T lymphocyte antigen 4-Ig, which blocks B7-mediated T-cell co-stimulation. CONCLUSIONS: Activation of peripheral organs following explosive brain death may be caused by various interrelated events, including the effects of massive acute central injury, hypotension, and circulating factors. Almost complete suppression of these changes could be produced by biological agents. Such interventions, if reproducible in humans, could improve the quality of organs from "marginal" donors, broadening the criteria for donor acceptance.

Mechanisms of indirect allorecognition: characterization of MHC class II allopeptide-specific T helper cell clones from animals undergoing acute allograft rejection.

BACKGROUND: Recent evidence indicates that T cells primed via the indirect pathway of allorecognition play an important role in allograft rejection, although the effector mechanisms remain unknown. The purpose of this study was to characterize and study the in vivo function of self-restricted MHC allopeptide-specific T-cell clones generated from animals undergoing allograft rejection. METHODS AND RESULTS: We generated self-restricted class II MHC allopeptide-specific T-cell clones from the spleen and kidney of Lewis (LEW; RT1l) rats undergoing acute rejection of MHC-incompatible Wistar Furth (WF; RT1u) renal allografts. RT1.Du/beta20-44 peptide-specific CD4+ T helper 1 clones from the spleen and kidney of rejecting animals expressed a restricted T cell receptor (TCR) Vbeta repertoire: Vbeta4, 8.2, or 9. In comparison, clones generated from RT1.Dubeta20-44 immunized LEW rats all expressed TCR Vbeta9. The amino acid sequence of RT1.Dl (LEW) and RT1.Du (WF) residues 20-44 differ only at positions 30 and 38. T-cell clones expressing TCR Vbeta9 preferentially proliferated to the peptide fragment RT1.Dubeta20-33. T-cell clones expressing TCR Vbeta4 proliferated weakly to peptide fragments RT1.Dubeta20-33 and 31-44, whereas those expressing TCR Vbeta8.2 proliferated preferentially to the peptide fragment 31-44. Adoptive transfer of T-cell clones expressing TCR Vbeta9 or Vbeta8.2, but not Vbeta4, to naive LEW animals elicited significant delayed-type hypersensitivity responses after challenge with the RT1.Dubeta20-44 peptide or allogeneic WF (RT1u) splenocytes. CONCLUSION: This is the first report on the cellular, molecular, and functional characterization of self-restricted MHC allopeptide-specific T-cell clones from animals undergoing acute rejection. Our data provide support for a biologically significant role of indirect allorecognition in allograft rejection.

Migration and activation pattern of specialized dendritic cells after heterotopic small bowel transplantation in a graft-versus-host model of the rat.

Besides specific cellular-mediated T cell responses, B cell related humoral responses have been demonstrated during the course of graft-versus-host disease after semiallogeneic transplantation of cellular antigen. Following semiallogeneic small bowel transplantation, there are, besides others, two specific forms of antigen-presenting cells, namely sinus lining cells (SLCs) and follicular dendritic cells (FDCs) which mediate primary and secondary humoral immune responses, respectively. This study was aimed to clarify the role of these dendritic cell entities after transplantation of small bowel grafts in a one-sided graft-versus-host (GvH) model for untreated and immunosuppressed (15-deoxyspergualin) recipient animals. As graft-versus-host disease progressed, SLCs and FDCs were eliminated in donor and recipient graft-versus-host associated target tissues (spleen and mesenteric lymph nodes) of untreated animals, whereas these dendritic cells prevailed in immunosuppressed recipients. 15-deoxyspergualin successfully prevented GvHD and significantly prolonged the mean survival time of untreated rats (16.0 +/- 4.5 d) for at least 21 d. Based on the immunosuppressive efficacy of 15-deoxyspergualin on the survival and function of SLCs and FDCs, an unaltered development of germinal centers and B cell proliferation within mesenteric lymph nodes and spleen was maintained

Influence of recipient pretreatment with donor spleen cells and 15-deoxyspergualin on rat skin graft survival.

Experimental studies performed in rat kidney transplantation showed that treatment with 15-deoxyspergualin (15-DOS) for 14 days after grafting may induce both permanent graft function and specific immune tolerance. The aim of this study was to check if donor spleen cell transfer and 15-DOS pretreatment before transplantation prolong skin graft survival. Pretreatment of the recipient with donor cells induced slight prolongation of subsequent skin graft survival. Addition of 15-DOS pretreatment for 14 days in a dose of 2.0 mg/kg b.w. to cell transfer did not influence survival of the graft. On the other hand, in the recipients receiving allogeneic spleen cells before transplantation and subtherapeutic doses of 15-DOS directly after grafting the prolongation of skin graft survival was observed. These results indicate that pretreatment of the recipients with donor cells before allogeneic skin transplantation allows for reduction of immunosuppression after grafting.

Mechanisms of indirect allorecognition in graft rejection: class II MHC allopeptide-specific T cell clones transfer delayed-type hypersensitivity responses in vivo.

Recent animal studies suggest that indirect T-cell recognition of alloantigen plays an important role in allograft rejection and tolerance. In this study, we generated T cell clones from Lewis (LEW, RT1(l)) rat lymph node cells that had been primed in vivo by immunization with immunogenic class II MHC allopeptide RT1.D(u)beta2, representing residues 20-44 of the polymorphic beta chain of RT1.Dbeta(u) (Wistar Furth [WF]). Using reverse transcriptase polymerase chain reaction transcript analysis with specific rat T cell receptor Vbeta primers, we show that six out of nine T cell clones specifically proliferated to RT1.D(u)beta2 and expressed Vbeta 9. One of these clones, clone 2F4, which specifically proliferated to RT1.D(u)beta2 in a dose-response fashion and produced interferon-gamma in response to restimulation by RT1.D(u)beta2, was selected for further studies. The beta-chains of RT1.D(l) and RT1.D(u) residues 20-44 differ by two amino acids at positions 30 and 38. We synthesized two peptides, each containing a single polymorphic site: RT1.D(u)beta 20-33 and RT1.D(u)beta 31-44. Both these peptides were immunogenic by LEW responders, since lymph node cells primed by immunization proliferated equally to the peptides in vitro. Interestingly, in vitro dose-response studies with clone 2F4 showed better proliferative response to peptide RT1.D(u)beta 20-33 than to peptide RT1.D(u)beta 31-44, indicating that this T cell clone preferentially recognizes a single amino acid difference on residue 30. Finally, it has been suggested that indirect allorecognition by CD4+ T cells mediate graft rejection by delayed-type hypersensitivity responses, although definitive studies are lacking. Systemic injection of the 2F4 clone to naive LEW rats elicited an antigen-specific delayed-type hypersensitivity response against RT1.D(u)beta2 peptide and WF splenocytes, confirming indirect presentation in vivo. These data demonstrate that Th1 cell clones generated by in vivo priming via the indirect pathway utilize specific T cell receptor Vbeta and recognize single amino acid differences in the allopeptide. More importantly,these Th1 clones are capable of mediating a specific immune response in vivo. These studies with MHC allopeptide-specific T cell clones further delineate the cellular mechanisms of indirect allorecognition and provide a potential strategy to study its role in acute and chronic rejection, and tolerance.

Analysis of tolerance inducing mechanism after application of oncogen-transformed macrophages.

The purpose of this study was to examine the expression of T cell receptors (TCR) and their V beta subclasses under the influence of the parental cell line P388D1 and its clones mos2 and mos3, using a mouse model. It was shown, that v-mos oncogene-transformed cells of this line (mos2) induced selective immunological unresponsiveness in vitro. Because the induction of tolerance is of a central importance for the organ transplantation, this phenomenon, found in vitro, was also studied in vivo. We found that the in vivo injection of mos2 cells into mice induced a state of selective noncreativity. To further analyse these effects, we studied whether specific tolerance is the consequence of a decreased number of essential receptors or receptor families. For this purpose C57BL/6 mice were immunized with cells of the parental line P388D1 or mos2 and mos3 clones. Their spleen and thymus cells were examined phenotypically. The most impressive result of this study was a clearly changed amount of T cells receptors in mos2 immunized mice, in which a state of tolerance was induced. In these mice only the expression of CD3 T receptors as well as that of the V beta 11 chains was reduced. In spleen of these mice the CD3 expression was decreased, compared to D1 or nonimmunized control animals by 54-58% and compared to mos3 mice by 38-40%. Even though the differences in the thymus were not very pronounced, we still saw a decrease in CD3 stained cells selective in mos2 immunized C57B1/6. The expression of V beta 11 chains on the surface of spleen cells of mos2 animals was reduced by 33.3%, on the thymocytes even by 50% comparing to that in nonimmunized mice. Whether the reduced expression of T receptor V beta families is due to changes in the genetic material (cDNA), has to be studied.

Induction of specific tolerance by 15-deoxyspergualin (DOS) treatment after rat skin and kidney transplantation in rats. Analysis of effectivity of various protocols of DOS application.

The purpose of this study was to analyse the effect of various protocols of 15-deoxyspergualin (DOS) application on skin or kidney graft survival. Following rat strain combinations were used: AS-->LEW (MHC identical/non-MHC-different) and DA-->LEW (MHC-different/non-MHC-different). Reference DOS dose was 2.5 mg/kg, i.p. It was shown that the effect of DOS depended on multiple factors, such as: type of tissue or organ, onset of treatment, drug dose and length of drug application. In skin transplantation graft survival was 32-34 days in AS-->LEW and 24-26 days in DA-->LEW. Kidney graft survived more than 150 days. DOS prolonged skin survival when the application was started earlier than day 8, whereas kidney graft survived only when DOS treatment was started not later than 3-4 days after transplantation. In skin transplantation a dose of 0.3 mg/kg had a small effect-prolongation graft survival up to 4 days. Higher doses induced longer graft survival, however, maximal survival of allogeneic skin was 22 days. In kidney transplantation a dose of 0.3 mg/kg led to prolonged graft survival-up to 150 days. Doses of 2.0-2.5 mg/kg were able to induce specific tolerance. The optimal skin or kidney graft survival was obtained when DOS was applied for 14 days. Shorter than 12-day treatment with DOS led to a shorter graft survival. When donor was pretreated with DOS prolongation of non-allogeneic graft survival was observed. Our results showed that short-term application of DOS is safe and effective. To obtain optimal DOS effect the drug application must be started directly after transplantation.

In vitro analysis of the mode of action of the immuno-suppressive drug 15-deoxyspergualin.

The purpose of the study was to analyse the action of the immunosuppressive drug 15-deoxyspergualin (DOS) in vitro. We studied: a) the influence of DOS alone and DOS in combination with various monoclonal antibodies on alloantigen stimulation in the mixed lymphocyte culture (MLC), b) the influence of DOS treatment on the MHC class I and II expression of splenocytes, lymph node cells and peritoneal macrophages, c) the influence of DOS treatment on a suppressor cell population. Our study showed that: a) DOS inhibits interleukin 1 (IL-1) secretion by macrophages, leading to reduction of immune response to alloantigens. This effect was neutralized by addition of IL-1; b) DOS treatment has no influence on MHC class II antigen expression, but induces changes of MHC class I expression. After DOS application in a population of spleen macrophages a subpopulation of cells with reduced MHC class I antigen expression appeared. Down-regulation of these molecules was also observed in immunomorphological studies of kidney graft sections of rats treated with DOS after transplantation; c) after DOS treatment suppressor cells were detected in "suppressor" MLC, 16-33 days after kidney transplantation. Their activity was confirmed 137 days after treatment with DOS, but were inactive in the case of third party cells. These results suggest that DOS action is based on a blockade of antigen presentation by reducing IL-1 production, down-regulation of MHC class I antigen and by inducing suppressor cell population.