Survival of macroencapsulated allogeneic parathyroid tissue one year after transplantation in nonimmunosuppressed humans.

The use of immunoisolation devices may allow transplantation without need for immunosuppression and could widen the indications for cell transplantation. In this study, we evaluated the survival of encapsulated parathyroid tissue in nonimmunosuppressed humans. Autologous parathyroid implants: Seven patients undergoing parathyroidectomy had devices containing small pieces of their own parathyroid tissue implanted SC. These devices were explanted after 2-4 weeks for histological evaluation. Allogeneic parathyroid implants: Four patients with chronic hypoparathyroidism were transplanted with one to three large (40 microl) and one small (4.5 microl) device filled with meshed parathyroid tissue and implanted SC. The small devices were explanted at 4 weeks, while the large ones were explanted 8.5 to 14 months after implantation. In both studies, control implants were placed in nude mice. Autologous study results: At explantation, the grafts consisted of 22 +/- 6% endocrine tissue and 63 +/- 7% fibrosis, while 15 +/- 5% of the grafts were necrotic. Allogeneic study results: In devices explanted from the patients at 4 weeks, fibrosis dominated and only 1%, 5%, and 23% of the grafts consisted of endocrine tissue. A similar histological appearance was found in grafts from nude mice. In devices explanted at 8.5-14 months, histologically intact endocrine tissue was found in all patients. However, nearly all the tissue consisted of fibrosis. There was no detectable increase in the parathormone (PTH) level in all patients. Macroencapsulated human allogeneic parathyroid tissue can survive up to 1 year after transplantation into nonimmunosuppressed patients. However, marked fibroblast overgrowth occurred, especially in the allogeneic implant study, using meshed parathyroid tissue. This was probably not related to the allo-response, because similar findings were observed in the nude mouse implants. In future studies, better tissue preparation and improvements in the physiological milieu inside the device may help to reduce fibroblast overgrowth and increase survival of the parathyroid cells.

Immunoisolation of tumor cells: generation of antitumor immunity through indirect presentation of antigen.

In recent years, a number of cancer therapies have been developed based on the augmentation of the immune response in the tumor-bearing individual. We have developed a novel approach to such therapies, using a membrane-bound immunoisolation device containing tumor cells to enhance the immune response of mice to the adenocarcinoma, MCA-38. Use of an immunoisolation device for cancer immunotherapy has several advantages. First, safety is enhanced because the reintroduced tumor cells are sequestered within the device. Second, the immunoisolation device permits introduction and maintenance of live cells that can stimulate the immune system for extended periods. Finally, the cells can be quantitatively removed at the end of the treatment period. Cells derived from the murine adenocarcinoma cell line MCA-38 were implanted into mice within a membrane-enclosed (immunoisolation) device. After 3 weeks, the animals were challenged by injection of free tumor cells. All of the control animals (lacking implants or with empty devices) developed tumor at the challenge site, whereas all of the animals that received devices containing tumor cells remained tumor free. The animals also remained tumor free after a second challenge. It is our hypothesis that antigens shed from tumor cells within the device are taken up by antigen-presenting cells of the host and thus indirectly initiate the activation of the immune system.

Variation in expression of porcine xenogeneic antigens.

The rejection of organs transplanted between phylogenetically disparate animals such as pigs and primates is thought to be initiated by binding of naturally occurring antibodies of the recipient to blood vessels in the donor organ. The major porcine target antigens are a triad of glycoproteins, gp115/135, which are expressed on endothelial cells and platelets of all animals tested to date. Whether expression of these antigens varies to the extent that selection of particular animals as donors for xenotransplantation would be warranted is an important question that has not been addressed previously. We tested the reactivity of human natural antibodies with platelet-derived antigens isolated from > 60 pigs representing 4 strains. Reactivity with IgM from a single human serum sample varied to such an extent as to suggest a 5-fold difference in the levels of antigen expression. Significantly, approximately 10% of animals express very low levels of the target antigens. The variations in antigen levels were not due to the age or sex of the animal or to environmental effects. There appears to be genetic regulation of the expression, as sibling pairs were more likely to have similar levels of reactivity than nonsiblings. Western blotting of platelet extracts shows the same quantitative differences in expression of the individual glycoproteins gp115, 125, and 135 as is seen by ELISA for all xenogeneic antigens. The levels of expression of gp115/135 appear to be controlled coordinately and are consistent with genetic regulation. While the clinical relevance of these observations is unclear, it is possible that lower levels of expression of these antigens on donor organs might lead to enhanced xenograft survival. In addition, in the experimental situation, the outcome of particular experiments might be affected by the selection of particular donor-recipient pairs.

Evidence that polyreactive antibodies are deposited in rejected discordant xenografts.

The rejection of organs transplanted between phylogenetically disparate species is thought to be initiated by the binding of naturally occurring antibodies of the recipient to the endothelium lining the blood vessels of the donor organ. We recently showed that among the xenoreactive natural antibodies in human serum that react with porcine endothelial cells and endothelial cell--derived glycoproteins are polyreactive antibodies. To test whether polyreactive natural antibodies are present in rejected xenografts we developed a series of hybridoma-derived antibodies specific for the polyreactive human monoclonal antibody 103, which we have shown to bind efficiently to porcine endothelial cells. Using these antiidiotypic reagents we detected antibodies bearing the 103 idiotype in a panel of human sera and on the surface of lymphocytes in the spleen of humans, rhesus monkeys, and baboons. The antiidiotypic reagents reacted in a pattern similar to the distribution of IgM, with immunoglobulin deposits in tissues obtained from pig-to-rhesus monkey and pig-to-baboon xenografts. Analysis of immunoglobulin eluted from these sites showed that they antibodies display the antigen-binding features of natural antibodies. Our findings are consistent with the hypothesis that idiotypes of polyreactive natural antibodies are broadly crossreactive. They also suggest that the polyreactive and xenoreactive IgM, which have been detected based on in vitro assays, may be deposited in a xenogeneic organ graft.

The natural immune barrier to xenotransplantation.

As the shortage of available organs for transplantation becomes critical, many investigators have turned to the possibility of using animals as a source of donor organs. Although there have been several attempts to use organs from closely related species for transplantation into human, there is relatively little experience in the use of nonprimate animals as clinical donor animals. The major problem in transplants between widely disparate species is hyperacute rejection, a rapid and violent rejection reaction that leads to the destruction of the graft within minutes or hours. Hyperacute rejection appears to be triggered by components of natural immunity, most notably natural antibodies and complement. Recent data suggest that hyperacute rejection may not represent an insurmountable barrier to discordant xenotransplantation. There have recently been several examples of survival of grafts in recipients in the face of antigraft antibodies and an intact complement system referred to as accommodation. Once hyperacute rejection can be averted, it becomes necessary to consider elicited cellular responses. There are a number of issues to be considered in the clinically relevant model of porcine to primate xenografts. These include the size of the responding T-cell repertoire and the extent to which the cell adhesion molecules and cytokines of the donor will be able to stimulate recipient immune responses. Finally, the interactions between T cells and the cells forming the inner layer of blood vessels may have profound effects on the outcome of the graft.

Gene expression in CD8 and CD4 T-cell populations following activation with the calcium ionophore A23187.

We have hypothesized that functional maturation of T lymphocytes can be dissected into a series of discrete stages. For example, activation of T lymphocytes with the calcium ionophore A23187 drives CD8+ T cells to become dividing blasts, referred to as 'pre-effector' cells in that these blasts do not express cytolytic function but are driven by IL-2 to do so. Here we characterize via Northern blots the functional maturation of CD8+ and CD4+ T-lymphocyte populations which have been activated via A23187 followed by stimulation with IL-2. Previously we have reported that no detectable IL-2 was found in the supernatants of A23187-activated pre-effector blasts. However, these cells do express levels of IL-2 mRNA very similar to those of OKT3-activated blasts, from which IL-2 is easily detected in the supernatant. Translational control may account for these findings. A23187-activated CD8+ pre-effector blasts do not respond to stimulation with IFN-gamma nor do they express IFN-gamma mRNA following stimulation with IL-2. These observations suggest that IL-2 may be sufficient to stimulate maturation of these cells. Activation via A23187 results in lower expression of the proto-oncogene c-myb relative to that found in OKT3 activation. C-myb mRNA levels are higher in CD8+ than in CD4+ A23187-activated pre-effector blasts and the c-myb level in the CD8+ pre-effector blasts falls in response to IL-2. This decrease in c-myb mRNA coincides with an increase in proliferation, and the expression of cytolytic activity.

Molecular events in late stages of T-cell functional maturation.

Peripheral blood lymphocytes activated with either the calcium ionophore A23187 or the combination of anti-CD2 monoclonal antibodies, 9.6 + VIT13, undergo blast formation and proliferation but do not develop cytolytic activity. These proliferating blasts, referred to as pre-effector blasts because they do not yet express cytolytic function, respond to stimulation with interleukin-2 (IL-2) by further proliferation and development of cytolytic activity, i.e. they become effector cells. Pre-effector blasts activated with 9.6 + VIT13, but not A23187-activated pre-effector blasts, also respond to stimulation with interferon-gamma (IFN-gamma) by becoming cytolytic effector cells. This report examines gene expression (by Northern blot analysis) in pre-effector blasts and during the transition from the pre-effector to the effector stage. The data presented here provide further support for the concept that A23187 activation drives T cells to become dividing blasts that are appropriately referred to as 'pre-effector' cells in that these blasts do not express transcripts for granzyme A or perforin mRNA but are driven by IL-2 to do so in parallel with the acquisition of cytotoxic function. Cells are apparently driven by 9.6 + VIT13 to a later stage of functional maturation than by A23187 activation; 9.6 + VIT13-activated pre-effector blasts express mRNA for both granzyme A and perforin, even though these blasts do not express cytolytic activity. Activation via A23187 results in lower expression of the proto-oncogene c-myb relative to that found in either 9.6 + VIT13 or OKT3-activated cells.

Role of cross-linking in stepwise activation of T cells.

Activation of T cells is a complex process which we have hypothesized involves a series of functional intermediates possessing some, but not all, of the characteristics of fully functional effector cells. We have identified two such functional intermediates, the poised T cell (poTc) and the pre-effector T cell (peTc). poTc do not proliferate or mediate cytolytic activity but are responsive to help in the form of interleukin 2 (IL-2); peTc are proliferating cells which are not cytolytic. Here the role of T-cell receptor/CD3 complex cross-linking in generating these functional intermediates is examined and it is shown that cells can be driven to different stages of functional maturation depending upon the sequence of antibody binding to CD3 and cross-linking. Highly purified T cells can be activated to poTc stage if they are first labelled with the anti-CD3 monoclonal antibody OKT3 and then cross-linked with goat anti-mouse IgG-coated beads. If the OKT3 antibody is first bound to the IgG-coated beads and then added to highly purified T cells, peTc are generated. In both cases the intermediates can be driven to become fully functional effector cells by the addition of IL-2. Finally, by removing the OKT3-bound beads during the activation process, were are able to show that poTc and peTc are sequential intermediates along the same pathway of activation.

The role of natural antibodies in the activation of xenogenic endothelial cells.

Hyperacute rejection of organ xenografts is thought to be mediated by the reaction of naturally occurring antibodies and complement of the recipient with blood vessels in the donor organ. We have suggested previously that the pathogenesis of hyperacute rejection might involve the activation of endothelial cells in the graft. To evaluate the potential role of natural antibodies and complement in hyperacute xenograft rejection, sixteen human sera were tested for variation in the ability to activate porcine endothelial cells as manifested by the release of biosynthetically labeled heparan sulfate from the cells. It was then asked to which extent such variation might reflect differences in natural antibody titer and/or complement activity. The sera mediated release of 3.6-57% of endothelial cell-associated heparan sulfate. Heparan sulfate release correlated significantly with the titer, in the sera, of IgM antibodies that bound to cultured endothelial cells (P = 0.0008) or to a triad of glycoproteins believed to represent the major targets of natural antibodies in porcine to primate xenografts (P = 0.001); correlation was also observed with the total concentration of IgM (P = 0.0046). The release of heparan sulfate did not correlate with corresponding properties of serum IgG, with anti-swine hemagglutination or with isohemagglutination titers. Heparan sulfate release correlated with deposition on endothelial cells of iC3b (P = 0.0095), but not with serum complement activity. Our findings indicate that in the reaction between human serum and xenogeneic endothelial cells, it is the concentration of xenoreactive IgM and not differences in complement activity that limits the ensuing pathophysiologic events.

Generation of lymphokine-activated killer activity in T cells. Possible regulatory circuits.

CD4+ and CD8+ T cells do not develop significant lymphokine-activated killer (LAK) activity when PBL are cultured with IL-2 or even when they are activated with a T cell stimulus such as OKT3 mAb. The possibility that a T cell regulatory mechanism prevents the development of LAK activity by CD4+ or CD8+ cells in OKT3 mAb and IL-2 cultures was tested by depleting CD8+ or CD4+ cells from PBL before stimulation with OKT3 and IL-2. Under these conditions, the remaining CD4+ and CD8+ cells were able to generate non-MHC-restricted lysis of NK-resistant tumor targets. Our data suggested that a regulatory signal was present in the culture to prevent the development of lytic function by T cells. T cells removed from the PBL cultures were, upon culture with IL-2, able to generate high LAK activity, suggesting that inhibition of the CD4+ or CD8+ T cell-mediated LAK activity was an active ongoing process, which blocked the lysis at the level of the activated cell and not the precursor cell. Mixing experiments demonstrated that the CD4+ or the CD8+ cells isolated from the PBL cultures were able to inhibit the development of lytic function in the CD4-depleted and CD8-depleted cultures. Transforming growth factor-beta (TGF-beta) has been shown to block LAK activity of NK cells in IL-2-stimulated cultures. When TGF-beta was added to CD4(+)- or CD8(+)-depleted cultures, it also inhibited LAK activity of T cells in a dose-dependent fashion, without interfering with T cell growth. Lytic activity returned to activated levels when TGF-beta was removed from the culture medium, thereby demonstrating the reversibility of TGF-beta inhibition.

Purified human T cells stimulated with cross-linked anti-CD3 monoclonal antibody OKT3: rIL-1 is a co-stimulatory factor for CD4+CD29+CD45RA- T cells.

Accessory cells (AC) are believed to play two major roles in T-cell activation: they cross-link certain stimuli such as monoclonal antibodies, and they provide needed cytokines. To differentiate between these roles, we cross-linked OKT3 on highly purified T cells by means of Fc-specific goat anti-mouse IgG-coated polystyrene beads and studied T-cell activation after exogenously added cytokines. Following addition of AC, rIL-2, or rIL-1, CD25 was up-regulated, and the cells proliferated and became cytotoxic. Both CD4+ and CD8+ cells were activated in the presence of AC or rIL-2. In contrast, only CD4+CD29+CD45RA- cells responded in the presence of rIL-1. Anti-IL-2R p55 (anti-TAC) monoclonal antibody inhibited the proliferative response supported by rIL-2 or rIL-1. To inhibit proliferation of cells stimulated in the presence of AC, anti-TAC needed to be supplemented with anti-IL-6 antibodies, or to be added in a 10-fold higher concentration. Cultures with AC produced larger amounts of IL-2 than those supplemented with rIL-1. Only AC-containing cultures also produced detectable amounts of IL-6. These findings combined with the observation that none of 2000 purified T cells counted in each of six independent experiments expressed MHC class II antigens strongly suggest that rIL-1 can activate T cells directly, rather than indirectly by potentiating the function of contaminating AC.