Prolonged survival of allogeneic islets in cynomolgus monkeys after short-term triple therapy.

Preclinical studies in nonhuman primates (NHP) are particularly useful to evaluate the safety and efficacy of new therapeutic proteins developed for use in clinical transplantation. We hypothesized that a treatment that selectively destroys activated cytopathic donor reactive T cells while sparing resting and immunoregulatory T cells in a mouse model might also produce long-term drug-free engraftment and tolerance without the hazards of lymphopenia in the challenging nonhuman primate islet allograft model. Short-term treatment with a regimen consisting of rapamycin, and IL-2.Ig plus mutant antagonist-type IL-15.Ig cytolytic fusion proteins (triple therapy) posttransplantation results in prolonged, drug-free engraftment of cynomolgus islet allografts. Moreover slow progressive loss of islet function in some recipients was not associated with obvious pathologic evidence of rejection.

Antiidiotypes against anti-H-2 monoclonal antibodies. V. In vivo antiidiotype treatment induces idiotype-specific helper T cells.

Mice have been treated in vivo with xenogeneic antiidiotypes prepared against a murine monoclonal anti-H-2Kk antibody, 11-4.1. B cell immune responses have been found to be altered by such treatment as evidenced by a modification in the idiotypic repertoire of the humoral response to H-2 antigens. Transfer of purified T cells into nude mice before anti-idiotype treatment showed that T cells are involved in the induction of idiotope-bearing antibodies by xenogeneic antiidiotype. Studies using bone marrow chimeras indicate that the environment in which either T or B cells mature does not appear to alter VH region genetic control of induction of antiidiotype-induced molecules. By adoptive transfer studies, T cells from antiidiotype-treated mice were found capable of modifying the idiotypic repertoire of B cells subsequently exposed to antigen even when the T cells were obtained from antiidiotype-primed mice of inappropriate allotype. Although it still must be determined whether idiotypic or antiidiotypic T cells are involved in such B cell idiotype regulation, these results indicate that some T cell functions are altered by xenogeneic antiidiotypes prepared against B cell products and suggest that T cell immunity to major histocompatibility complex antigens may also be affected by such reagents.

Xenogeneic skin graft rejection is especially dependent on CD4+ T cells.

B6 mice were treated in vivo with anti-CD4, anti-CD8, or both anti-T cell antibodies together in an effort to prolong xenogeneic compared with allogeneic skin graft survival. Mice treated with anti-CD4 antibody showed prolonged survival of xenogeneic monkey or rabbit skin even after they had rejected whole MHC-disparate allogeneic mouse skin. Furthermore, the addition of cyclosporine was synergistic with the anti-CD4 antibody in prolonging graft survival. These results suggest that the cell-mediated response to xenogeneic antigens is especially dependent on CD4+ lymphocytes, a feature shared by the response to allogeneic minor histocompatibility antigens. In addition, the results suggest a possible approach to clinical immunosuppression for some forms of xenogeneic transplantation.

Xenogeneic proliferation and lymphokine production are dependent on CD4+ helper T cells and self antigen-presenting cells in the mouse.

We studied proliferation and interleukin 2 production by B6 mouse spleen cells in response to stimulation by irradiated cynomolgus monkey spleen cells and compared the results with responses against whole MHC-disparate allogeneic controls (BALB/c). We found that (a) primary xenogeneic helper responses were absent, whereas primary allogeneic responses were brisk, (b) secondary xenogeneic helper responses were dependent on CD4+ T cells and responder antigen-presenting cells (APCs), whereas allogeneic responses could be mediated by either CD4+ or CD8+ T cells independently and were primarily dependent on the presence of stimulator APCs, and (c) secondary xenogeneic helper responses were blocked by an antibody directed against responder class II MHC molecules. These results suggest that mouse helper T cells recognize disparate xenoantigens as processed peptides in association with self class II MHC molecules, similar to the recognition of nominal antigens and unlike direct allo-recognition.

Indirect recognition by helper cells can induce donor-specific cytotoxic T lymphocytes in vivo.

In vitro studies have revealed that help for cytotoxic T lymphocyte (CTL) induction can be mediated through several pathways, including direct recognition of allogenic class II antigens by CD4+ cells, direct recognition of allogeneic class I antigens by "CD4-independent" CD8+ cells, and "indirect" recognition of peptides of alloantigens presented in association with self class II molecules. Whereas good evidence for the two direct pathways is available in vivo, there is relatively little evidence to show that indirect recognition can initiate graft rejection. This study examined the role of indirect allorecognition during the generation of CTLs in mice as they rejected major histocompatibility complex (MHC) class II-deficient skin after depletion of CD8+ T cells in vivo. Recipients were depleted of CD8+ T cells by in vivo treatment with anti-CD8 monoclonal antibody and then grafted with allogeneic skin lacking MHC class II antigens. The mice rejected the skin grafts rapidly. Although flow cytometry showed marked depletion of CD8+ T cells in these mice, we found that (a) CD8+ CTLs were generated and sensitized to MHC class I antigens of the donor; (b) the generation of the CD8+ CTLs required the help in vivo of CD4+ cells, as well as priming with the allogeneic skin graft; and (c) the CD4+ T helper cells were sensitized indirectly to donor peptides presented in association with class II antigens on recipient antigen-presenting cells. These results provide evidence that indirect recognition can provide effective help for CTL induction during graft rejection, even when the cytotoxic T cells are sensitized by determinants expressed only on the donor graft.

Skin graft rejection by beta 2-microglobulin-deficient mice.

Mice homozygous for a beta 2-microglobulin (beta 2-m) gene disruption lack beta 2-m protein and are deficient for functional major histocompatibility complex class I (MHC-I) molecules. The mutant mice have normal numbers of CD4+8- T helper cells, but lack MHC-I-directed CD4-8+ cytotoxic T lymphocytes (CTLs). In this study we used the beta 2-m mutant mice to study the importance of MHC-I-directed immunity in skin graft rejection. Our results indicate that MHC-I-directed CD8+ CTLs are not essential in the rejection of allografts with whole MHC or multiple minor H differences. However, the absence of MHC-I-guided immunity profoundly reduces the ability of mutant mice to reject H-Y disparate grafts. In addition, we show that natural killer cells which vigorously reject MHC-I-deficient bone marrow grafts, are not effective in the destruction of MHC-I-deficient skin grafts.

Two levels of help for B cell alloantibody production.

We have examined whether T cell stimulation by direct or indirect pathways contributes to alloantibody production by B cells after major histocompatibility complex (MHC)-disparate skin graft rejection in mice. Experiments were performed using normal mice, MHC class II-deficient mice, MHC class II-deficient mice with an intact peripheral CD4+ cell population (due to expression of class II antigens only on thymic epithelium), mice lacking the cytoplasmic tail of their MHC class II antigens, and mice depleted of CD4+ cells by anti-CD4 monoclonal antibody treatment. Depletion of recipient CD4+ cells reduced alloantibody production to barely detectable levels. Absence of donor MHC class II antigens did not affect the production of either immunoglobulin (Ig)M or IgG antibodies directed at class I alloantigens. Absence of recipient MHC class II antigens, however, led to production of only IgM but not IgG antibodies, even if the recipients had an intact CD4+ cell population. Absence of the cytoplasmic tail of the recipient's MHC class II antigens led to the production of slightly reduced amounts of IgG antibody. These findings indicate that (a) CD4+ cells are essential helper cells for B cell alloantibody production; (b) production of IgM alloantibody can occur with help from CD4+ cells, which recognize either donor class II antigens or modified recipient class II antigens; (c) isotype switching from IgM to IgG alloantibody requires help from CD4+ cells activated by antigens presented by recipient MHC class II molecules; and (d) the cytoplasmic domain of the recipient MHC class II molecules may be involved in the mechanism that leads to isotype switching by B cells. Thus, there are two levels of CD4-mediated help available for B cells responding to alloantigens: one (involving a noncognate interaction) can produce B cell activation, and a second (involving a cognate interaction) is required for differentiation and IgG alloantibody production.

Effect of class II antigen matching on renal allograft survival in miniature swine.

The benefit of class II major histocompatibility complex (MHC) antigen matching to renal allograft survival, in the absence of immunosuppression, has been studied in partially inbred miniature swine. Permanent (greater than 6 mo) renal allograft survival was found in 30% of recipients of either class II only or fully matched grafts. Analysis of the survival of the class II-only matched grafts by specific recipient/donor haplotype combinations indicated that survival was regulated by at least three genetic factors, including antigen gene dose, a class I MHC allele-dependent effect, and non-MHC-linked immune response phenomenon. Animals accepting class II-matched kidneys developed spontaneous tolerance to the graft, despite mounting an initial immune response marked by renal damage and the development of serum cytotoxic antibodies directed at the donor MHC antigens. The antibodies were only of the IgM class, suggesting that conversion of the humoral response to IgG was blocked. After acceptance of the kidney, three out of five animals showed specific prolongation of donor skin grafts. At the time of rejection of these skin grafts, no decrease in renal function nor reappearance of anti-donor antibodies was observed.

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.

Cellular mechanisms of rejection.

Studies by molecular biologists, protein chemists and cell biologists are rapidly providing new tools and information for those interested in the cellular mechanisms of graft rejection. Despite these contributions, a clear picture of the mechanisms involved in rejection has not yet evolved. However, whole new areas for research have developed, providing opportunities for new insights as well as therapeutic interventions.

Antigen processing and presentation in transplantation.

For many years the direct stimulation of T cells in response to donor MHC antigens expressed on donor antigen-presenting cells has been the focus of transplantation immunology. Indirect recognition in response to peptides of donor antigens presented by self MHC molecules on recipient antigen-presenting cells has not generally been considered an important feature of graft rejection. Recent evidence suggests that indirect responses may be more important than previously considered and the new emphasis on indirect pathways in allograft rejection has raised new issues, many of which are unresolved.

Mechanisms of tolerance in murine radiation bone marrow chimeras. II. Absence of nonspecific suppression in mature chimeras.

Spleen cells from a series of allogeneic bone marrow chimeras were sensitized in vitro with stimulator cells from major histocompatibility complex recombinant strains of mice. The combinations were chosen such that both tolerated (host or donor) and nontolerated (third-party) antigens were present on the same stimulator cells in order to determine whether the tolerated host antigens might elicit nonspecific suppressor mechanisms affecting the cell-mediated lympholysis (CML) response to the nontolerated antigens. No evidence for such nonspecific suppression was obtained in several types of assays. Therefore, if suppressor mechanisms exist that mediate such tolerance in mature allogeneic chimeras then these mechanisms must be highly antigen-specific.

Mechanisms of tolerance in murine radiation bone marrow chimeras. I. Nonspecific suppression of alloreactivity by spleen cells from early, but not late, chimeras.

Allogeneic chimeras were prepared using lethally irradiated B6 hosts and untreated marrow from exsanguinated BALB/c donors. For about two months after reconstitution, chimeras had very weak antihost cell-mediated lymphocytotoxicity (CML) reactivity and little third-party alloreactivity. During this time a cell population capable of suppressing CML reactivity against both host and third-party alloantigens (i.e., antigen-nonspecific) was demonstrated in chimera spleens by in vitro mixing experiments. The putative suppressor cells were Thy-1-negative and radiation-sensitive. Subsequently, mature chimeras showed host tolerance and strong third-party alloreactivity. At this point suppressor mechanisms could no longer be demonstrated. These data are consistent with a clonal elimination hypothesis in that they do not provide evidence to indicate that maintenance of specific immune tolerance is mediated by an active suppressor mechanism.

Evidence that multiple defects in cell-surface molecule interactions across species differences are responsible for diminished xenogeneic T cell responses.

The purpose of the present study was to identify which of the several possible defects in cell-surface-molecule interactions are responsible for diminished mouse helper T cell responses to xenoantigens. We measured primary mouse anti-monkey, anti-pig, and anti-human proliferation in vitro in experimental systems in which potential defects were partially corrected by lymphokine supplementation and/or the use of transgenic or hybridoma cell populations. We found that the diminished mouse helper T cell responses to xenoantigens result from at least two defects in cell-surface-molecule interactions between T cells and xenogeneic APCs, specifically TCR and/or CD8 interactions with xenogeneic class I MHC molecules and accessory molecule interactions with their ligands (probably LFA-1 with ICAM-1/ICAM-2 and/or LFA-2 with LFA-3). Other investigators have identified additional defects, such as in lymphokine function across species differences. Thus, there appear to be multiple defects responsible for the diminished cellular immune response to xenoantigens.

Prevention of alloantibody formation after skin grafting without prolongation of graft survival by anti-L3T4 in vivo.

Treatment of mice in vivo with monoclonal antibodies against the L3T4 antigen (CD4 in human beings) has been shown to suppress the humoral response to several foreign antigens and to prolong the survival of allografts in some cases. Experiments were therefore performed to test whether anti-L3T4 antibody treatment would suppress alloantibody production after skin transplantation. Monoclonal anti-L3T4 antibody (GK1.5) was administered to C57BL/6 (B6) mice prior to BALB/c skin grafting. The production of B6 anti-BALB/c alloantibody was then tested after graft rejection. The results showed that: (1) graft survival of BALB/c skin on B6 mice was not substantially prolonged by anti-L3T4 treatment; (2) graft survival was significantly prolonged if mice were treated with both anti-L3T4 and anti-Lyt2 antibody; (3) the production of alloantibody following grafting was decreased by anti-L3T4 treatment and was completely eliminated if thymectomy was also performed; (4) thymectomy prolonged the effectiveness of the anti-L3T4 treatment; (5) tolerance to alloantigens presented at the time of anti-L3T4 treatment was not achieved; and (6) well-established cytotoxic antibody titers rose to higher levels after secondary grafting even with concurrent anti-L3T4 treatment, while weak antibody titers remained stable or decreased. These results indicate that L3T4+ cells are essential in providing the "help" necessary for generating humoral responses to alloantigens but that elimination of these L3T4+ cells still allows the generation of help for cell-mediated immunity. The data also suggest that class I antigens must be presented on class II molecules in order to elicit an antibody response.

T cell recognition of xeno-MHC peptides during concordant xenograft rejection.

T cell recognition of xenoantigens is likely to play a key role in rejection of xenografts surviving hyperacute and delayed xenograft rejection, but the mechanisms of how this might occur are unknown. We used synthetic rat class II MHC peptides to study the role of the indirect pathway, where processed xenogeneic MHC antigens are presented in the context of self MHC, in a concordant xenograft rejection model in vivo. Mice of four different strains, BALB/c, B1O.A, CBA/ca, and C57BL/6j, were immunized with a mixture of rat class II MHC 25-mer xenopeptides representing the full-length sequence of the beta chain hypervariable domain of either RT1.Du (DR and I-E like) or RT1.Bu (DQ and I-A like) of the Wistar-Furth (WF) (RT1u) rat. Draining lymph node cells were capable of recognizing and proliferating to specific class II xeno-MHC peptides. The immunogenicity of the different peptides varied with the responder mouse strain. Responder T cells were CD4+, and were inhibited by anti-I-A and anti-I-E antibodies. We then examined the proliferative response of T cells from B1O.A primed by WF skin or vascularized cardiac xenografts to the class II MHC xenopeptides, when presented by naive B1O.A splenic antigen-presenting cells. These T cells were capable of proliferating to the same xeno-MHC peptides shown to be immunogenic by immunization. These data confirm the occurrence of self-restricted T cell recognition of xeno-MHC peptides in xenograft rejection, and provide the rationale for further investigating the role of the indirect pathway of recognition in xenotransplantation.

Delayed rejection of soluble tumor necrosis factor receptor-secreting tumor allografts.

BACKGROUND: Exogenous soluble tumor necrosis factor receptor (TNFR) has been shown to be an effective immunosuppressant. It has yet to be tested whether tissues secreting soluble TNFR, when transplanted into a foreign host, could locally generate immunosuppression and therefore manifest prolonged survival. METHODS: A murine tumor line was transfected with the gene encoding a chimeric protein consisting of the extracellular domain of the human 75-kDa TNFR fused to the Fc region of the human IgG1 heavy chain. This tumor line was then injected into allogeneic recipients. RESULTS: Transfected tumor cells were shown to secrete soluble TNFR. When transplanted into minor histocompatibility antigen-disparate allogeneic recipients, these tumor cells grew as a solid tumor and resisted rejection, whereas untransfected tumors and interleukin-4 receptor transfectant controls were rejected within 4 weeks. The resistance to rejection could be reversed by coadministration of an anti-TNFR monoclonal antibody. CONCLUSIONS: Prolongation of graft survival can be achieved by genetically altering transplanted tissue to secrete soluble cytokine receptors.

CD8+ effector cells responding to residual class I antigens, with help from CD4+ cells stimulated indirectly, cause rejection of "major histocompatibility complex-deficient" skin grafts.

BACKGROUND: Skin grafts from mice that are deficient in the expression of both class I and class II major histocompatibility complex (MHC) antigens are rejected rapidly by normal recipients. METHODS: To determine the mechanism of this rejection, MHC-deficient skin grafts were placed on recipients with different degrees of antigenic disparity and on recipients depleted of selected T cell subpopulations. In addition, the recipient's T cells were examined in vitro for their responses before and after graft rejection. RESULTS: The results indicate that (1) CD4+ cells provide help for this rejection by recognizing donor antigens presented by recipient class II antigens, and (2) CD8+ cells can participate as effector cells, recognizing residual class I antigens expressed by the MHC-deficient grafts. CONCLUSIONS: The primary conclusion from these studies is that the supposedly MHC-deficient mice actually do have sufficient class I antigen expression to cause skin graft rejection. This finding prevents the use of these mice to answer definitively the question of whether grafts entirely lacking MHC antigens would be rejected. However, these studies do illustrate two important (although previously recognized) features of allogeneic skin graft rejection: (1) that rejection can be initiated by help provided entirely through the indirect pathway, and (2) that help provided through the indirect pathway is available for effector T cells sensitized directly by donor cells. However, the results from these and other studies suggest that indirect effector mechanisms would probably be able to destroy truly MHC-deficient grafts under some circumstances.

Long-term metabolic and quality of life results with pancreatic/renal transplantation in insulin-dependent diabetes mellitus.

Evaluation of whole-organ pancreas transplantation in the therapy of IDDM has been difficult because of generally poor graft survival and significant complications in past experience. We report a technically successful simultaneous pancreas/kidney transplant program with patient and graft survival of 85% over 3 years of follow-up (mean 21 months) in 33 subjects with IDDM. Glucose metabolism was normalized without need for exogenous insulin immediately posttransplant in all but one recipient and remained normal in 85% of recipients. The outcome in pancreas/kidney recipients was compared with that in 18 insulin-dependent diabetic recipients of kidney transplant only performed in the same period. Quality of life was assessed with one general and one diabetes-specific questionnaire. General quality of life issues improved significantly in both pancreas/kidney and kidney recipients, but diabetes specific quality of life improved only in the pancreas/kidney recipients. Pancreas/kidney recipients required twice as long a period of hospitalization for the transplant and two times as many readmissions for a variety of complications. Only a minority of hospital admissions was strictly attributable to the pancreas graft. Of the five deaths in the pancreas/kidney recipients, two were attributable to the pancreas transplant. Pancreas transplantation in IDDM can now be accomplished with a high degree of success, resulting in normalized glucose metabolism and with overall mortality similar to kidney transplantation alone. Successful pancreas transplantation improves quality of life with respect to diabetes but this benefit is accomplished at a cost of increased hospital admissions and complications related to the transplanted pancreas. The effects of pancreas transplantation on the long-term complications of insulin-dependent diabetes remain unknown.

Liver transplantation for primary hepatic cancer.

Although early survival following transplantation for primary hepatic cancer is excellent, previously reported high recurrence rates have generally discouraged liver replacement for this indication. Since the inception of the Boston Center for Liver Transplantation (BCLT) in 1983, 33 of 383 (8.6%) liver allograft recipients have undergone orthotopic transplantation as definitive treatment for otherwise unresectable cancer. Diagnoses included hepatocellular carcinoma (HCCA) in 24 patients (73%), and cholangiocarcinoma (CHCA) in 9 patients (27%). Actuarial survival rates for patients with hepatocellular carcinoma were 71%, 56%, and 42% at 1, 2, and 3 years, respectively. The actuarial survival rates for patients with cholangiocarcinoma were 89% at 6 months, and 56% at 1, 2, and 3 years. Of the nine patients with cholangiocarcinoma, 56% (5/9) developed recurrent disease. Although this recurrence rate is disheartening, because of the lack of other morbidity, long-term survival in these patients is comparable to patients with HCCA. In contrast, recurrent hepatocellular carcinoma developed in 25% of recipients (5/20) who survived longer than 3 months posttransplantation. Other causes of death in patients with hepatocellular carcinoma included perioperative complications, 16.6% (4/24); sepsis, 8.3% (2/24); coronary artery disease, 4.2% (1/24); and lymphoma, 4.2% (1/24). Favorable prognostic factors included: primary tumor less than 3 cm in size and absence of associated cirrhosis. These results emphasize that orthotopic liver transplantation can provide a long-term cure for approximately 50% of patients whose primary hepatic malignancy is unresectable by conventional procedures.