Approaches to avoid immune responses induced by repeated subcutaneous injections of allogeneic umbilical cord tissue-derived cells.

BACKGROUND: Cellular treatments for repairing diseased tissues represent a promising clinical strategy. Umbilical cord tissue-derived cells (UTC) are a unique source of cells with a low immunogenic profile and potential for tissue repair. By using UTC from miniature swine, we previously demonstrated that despite their low immunogenic phenotype, UTC could induce an immune response under certain inflammatory conditions and after multiple subcutaneous (SC) injections. Given that repeat dosing of cells may be necessary to achieve a lasting therapeutic benefit, in this study, we examined approaches to avoid an immune response to multiple SC injections of UTC. METHODS: By using in vitro and in vivo measures of sensitization to SC cellular injections, we assessed the effects of varying the location of administration site, prolongation of timing between injections, and use of immunosuppressive treatments on repeated cellular injections in Massachusetts General Hospital major histocompatibility complex-defined miniature swine. RESULTS: Although under normal conditions, a single SC injection of major histocompatibility complex-mismatched UTC did not induce a detectable immune response, multiple SC injections of UTC demonstrated rapid humoral and cell-mediated immune responses. Avoidance of an immune response to repeat SC injection was achieved by concurrent immunosuppression with each dose of UTC. CONCLUSIONS: UTC and other similar cell types believed to be nonimmunogenic have the potential to induce immune responses under certain conditions. These studies provide important considerations and guidelines for preclinical studies investigating allogeneic cellular therapies.

Immunogenicity of umbilical cord tissue derived cells.

Umbilical cord tissue provides a unique source of cells with potential for tissue repair. Umbilical cord tissue-derived cells (UTCs) are MHC class I (MHCI) dull and negative for MHC class II (MHCII), but can be activated to increase MHCI and to express MHCII with IFN-gamma stimulation. Mesenchymal stem cells with similar characteristics have been inferred to be nonimmunogenic; however, in most cases, immunogenicity was not directly assessed. Using UTC from Massachusetts General Hospital MHC-defined miniature swine, we assessed immunogenicity across a full MHC barrier. Immunogenicity was assessed by in vitro assays including mixed lymphocyte reaction (MLR) and flow cytometry to detect serum alloantibody. A single injection of MHC-mismatched unactivated UTCs did not induce a detectable immune response. When injected in an inflamed region, injected repeatedly in the same region or stimulated with IFN-gamma prior to injection, UTCs were immunogenic. As clinical cellular repair strategies may involve injection of allogeneic cells into inflamed regions of damaged tissue or repeated doses of cells to achieve the desired benefit, our results on the immunogenicity of these cells in these circumstances may have important implications for optimal success and functional improvement for this cellular treatment strategy for diseased tissues.

Anti-CD3 antibodies OKT3 and hOKT3gamma1(Ala-Ala) induce proliferation of T cells but impair expansion of alloreactive T cells; aspecifc T cell proliferation induced by anti-CD3 antibodies correlates with impaired expansion of alloreactive T cells.

OKT3, a mouse anti-human CD3 monoclonal antibody (mAb), has been used for decades to reverse acute transplant rejection. A humanized OKT3 with two alanine replacements on the CH2 domain, hOKT3gamma1(Ala-Ala), was generated to reduce side effects of OKT3. This study reports the effects of OKT3 and hOKT3gamma1(Ala-Ala) on responder T cells in mixed leukocyte cultures (MLC). T cells were purified from peripheral blood mononuclear cells (PBMC) and labeled with CFSE before culture with irradiated allogeneic PBMC in a MLC. Multiparameter flow cytometric analysis of MLC demonstrated logarithmic expansion of T cells with a memory phenotype. Treatment with either OKT3 or OKT3gamma1(Ala-Ala) prevented the logarithmic expansion of these cells. Instead, both OKT3 and hOKT3gamma1(Ala-Ala) induced nonspecific expansion of T cells with both naive and memory phenotype. The proliferating T cells in OKT3 or hOKT3gamma1(Ala-Ala) treated cultures expressed low levels of CD25, and showed a diminished Granzyme B expression compared to that of the untreated MLC, suggesting that these cells may not be fully functional. An array of cytokines in the MLC supernatant was analyzed, and IL-5 and IL-13 were significantly reduced by OKT3 or hOKT3gamma1(Ala-Ala) treatment. These results demonstrated that OKT3 and hOKT3gamma1(Ala-Ala) have similar effects on T cells in MLC. Both antibodies inhibited the logarithmic expansion of allo-reactive T cells, probably through inducing suboptimal proliferation of specific and non-specific T cells. This in vitro study provided one possible mechanism of the therapeutic effects of OKT3 and hOKT3gamma1(Ala-Ala) antibodies.

Vascular endothelium does not activate CD4+ direct allorecognition in graft rejection.

Expression of MHC class II by donor-derived APCs has been shown to be important for allograft rejection. It remains controversial, however, whether nonhemopoietic cells, such as vascular endothelium, possess Ag-presenting capacity to activate alloreactive CD4(+) T lymphocytes. This issue is important in transplantation, because, unlike hemopoietic APCs, allogeneic vascular endothelium remains present for the life of the organ. In this study we report that cytokine-activated vascular endothelial cells are poor APCs for allogeneic CD4(+) T lymphocytes in vitro and in vivo despite surface expression of MHC class II. Our in vitro observations were extended to an in vivo model of allograft rejection. We have separated the allostimulatory capacity of endothelium from that of hemopoietic APCs by using bone marrow chimeras. Hearts that express MHC class II on hemopoietic APCs are acutely rejected in a mean of 7 days regardless of the expression of MHC class II on graft endothelium. Alternatively, hearts that lack MHC class II on hemopoietic APCs are acutely rejected at a significantly delayed tempo regardless of the expression of MHC class II on graft endothelium. Our data suggest that vascular endothelium does not play an important role in CD4(+) direct allorecognition and thus does not contribute to the vigor of acute rejection.

Flow cytometric test for beryllium sensitivity.

BACKGROUND: Chronic beryllium disease (CBD) is an occupational granulomatous disorder characterized by hypersensitivity to beryllium, mediated by CD4+ T lymphocytes, and predominantly affects the lungs. In this disorder, lymphocyte proliferative responses to beryllium, measured by 3H thymidine incorporation, are used for diagnosis of CBD, for screening asymptomatic workers or former workers to detect unrecognized disease, and for surveillance as a bioassay to detect abnormal exposures. Problems with test variability and the use of radioactivity have recently led to the search for alternative methods. METHODS: We applied a 5,6-carboxyfluorescein diacetate succinimidyl ester flow cytometric technique for measurement of mitogen- and antigen-induced T-lymphocyte proliferation to a group of beryllium-exposed sensitized individuals and beryllium-unexposed controls. RESULTS: We detected mitogen and antigen proliferative responses in CD3+, CD4+, and CD8+ subpopulations. Phytohemagglutinin and Candida stimulated CD4+ and CD8+ T-cell responses, but beryllium appeared to stimulate only CD3+/CD4+ responses. CONCLUSIONS: This technique may provide a sensitive, nonradioactive alternative to the traditional proliferation tests that measure beryllium sensitivity. It offers the added specificity of enabling phenotypic description of the responding cell type and may prove to be easier to standardize for clinical use.

Mouse vascular endothelium activates CD8+ T lymphocytes in a B7-dependent fashion.

Despite several studies examining the contribution of allorecognition pathways to acute and chronic rejection of vascularized murine allografts, little data describing activation of alloreactive T cells by mouse vascular endothelium exist. We have used primary cultures of resting or IFN-gamma-activated C57BL/6 (H-2(b)) vascular endothelial cells as stimulators and CD8(+) T lymphocytes isolated from CBA/J (H-2(k)) mice as responders. Resting endothelium expressed low levels of MHC class I, which was markedly up-regulated after activation with IFN-gamma. It also expressed moderate levels of CD80 at a resting state and after activation. Both resting and activated endothelium were able to induce proliferation of unprimed CD8(+) T lymphocytes, with proliferation noted at earlier time points after coculture with activated endothelium. Activated endothelium was also able to induce proliferation of CD44(low) naive CD8(+) T lymphocytes. Activated CD8(+) T lymphocytes had the ability to produce IFN-gamma and IL-2, acquired an effector phenotype, and showed up-regulation of the antiapoptotic protein Bcl-x(L). Treatment with CTLA4-Ig led to marked reduction of T cell proliferation and a decrease in expression of Bcl-x(L). Moreover, we demonstrate that nonhemopoietic cells such as vascular endothelium induce proliferation of CD8(+) T lymphocytes in a B7-dependent fashion in vivo. These results suggest that vascular endothelium can act as an APC for CD8(+) direct allorecognition and may, therefore, play an important role in regulating immune processes of allograft rejection.

Mechanism of T cell-mediated endothelial apoptosis.

BACKGROUND: Cytotoxic T lymphocyte (CTL)-mediated destruction of allogeneic vascular endothelium is important in the pathogenesis of both acute and chronic allograft rejection. Despite the importance of this phenomenon, the effector mechanisms responsible for endothelial cell killing are not well defined, and conflicting conclusions have been reached based on variation in experimental methodology. METHODS: We used a recently described method for isolating mouse vascular endothelium to evaluate endothelial cell lysis by CTLs. Endothelial cell destruction was assessed in vitro both by 51Cr release and DNA fragmentation using wild-type and lpr (Fas deficient) endothelium of C3H/HeJ (H2(k)) mice by MHC alloantigen-specific T cells of wild-type, gld (Fas ligand deficient), and perforin-deficient mice on a C57BL/6 (H2(b)) background. RESULTS: Although maximal lysis of 56.6+/-0.8% was seen when using wild-type targets and effectors, only a moderate decrease in apoptosis to 37.6+/-4.0% was detected when the Fas/Fas ligand death receptor pathway was eliminated. This decrease in cytotoxicity occurred despite the preserved functional capacity of this pathway. Alternatively, a significant decrease in cytotoxicity to 17.4+/-4.7% was seen when the perforin/granzyme exocytosis pathway was eliminated. CONCLUSIONS: These data indicate that CTLs destroy vascular endothelium primarily by the perforin/granzyme exocytosis pathway with only a minor contribution to apoptosis by the Fas/Fas ligand death receptor pathway. These data are critical for the proper interpretation of studies evaluating acute and chronic allograft rejection and for the design of rational strategies to ameliorate vascular injury concomitant to the rejection process.

The role of passenger leukocyte genotype in rejection and acceptance of rat liver allografts.

BACKGROUND: Although graft-resident passenger leukocytes are known to mediate acute rejection by triggering direct allorecognition, they may also act in an immunomodulatory fashion and play an important role in tolerance induction. Our purpose in the current study was to utilize rat bone marrow chimeras to evaluate the role of the genotype of passenger leukocytes in both acute rejection and tolerance of liver allografts. METHODS: The fate of livers bearing donor-type, recipient-type, and third-party passenger leukocytes was evaluated in the MHC class I and II mismatched rejector combination ACI-->LEW and the acceptor combination PVG-->DA. RESULTS: We report that although treatment of ACI liver donors with lethal irradiation does not lead to prolongation of graft survival in the ACI-->LEW strain combination, ACI livers bearing recipient-type (LEW) or third-party passenger leukocytes (BN) are rejected at a significantly slower rate. We confirm that lethal irradiation of PVG donor animals leads to abrogation of tolerance induction with acute rejection of their livers by DA recipients. However, the majority of PVG livers carrying donor-type (PVG), recipient-type (DA), or third-party (LEW) passenger leukocytes are accepted for >100 days. These DA recipients develop immune tolerance to the donor parenchyma (PVG). CONCLUSIONS: Our findings demonstrate that long-term acceptance of liver allografts and tolerance induction is not dependent on the presence of donor-type passenger leukocytes and can be achieved with organs carrying donor-type, recipient-type, or third-party passenger leukocytes. The importance of the MHC framework on the surface of passenger leukocytes as a critical regulator of the immune response after transplantation of chimeric organs is substantiated by the delayed tempo of rejection of ACI livers bearing recipient-type or third-party passenger leukocytes in the ACI-->LEW strain combination.

Depletion of recipient CD4+ but not CD8+ T lymphocytes prevents the development of cardiac allograft vasculopathy.

BACKGROUND: We have described that chimeric rat hearts bearing recipient-type antigen-presenting cells (APCs) do not reject acutely, but develop cardiac allograft vasculopathy (CAV) in untreated recipients. This suggests that CAV is triggered either by CD8+ direct allorecognition or by CD4+ indirect allorecognition. To determine the allorecognition pathway responsible for CAV in this model, recipients of chimeric hearts underwent either CD8+ or CD4+ T cell depletion. METHODS: Chimeric hearts were created via bone marrow transplantation in two fully major histocompatibility-mismatched rat strain combinations. DA recipients were thymectomized and treated with Ox8 and Ox38 murine monoclonal antibodies, which deplete CD8+ and CD4+ T cells, respectively. Chimeric PVG hearts bearing DA APCs, abbreviated PVG(DA), were heterotopically transplanted into recipients undergoing thymectomy alone or recipients undergoing thymectomy plus either CD4+ or CD8+ T cell depletion. RESULTS: PVG(DA) allografts survived 100 days, but developed CAV in thymectomized recipients and in those permanently depleted of CD8+ T cells. In contrast, chimeric hearts transplanted into permanently CD4+ T cell-depleted recipients survived 100 days and demonstrated no evidence of CAV. CONCLUSIONS: In this specific strain combination, recipient CD8+ T cells are neither necessary nor sufficient for the development of CAV, whereas recipient CD4+ T cells are required for the development of CAV. These findings suggest that CAV is dependent on CD4+ indirect allorecognition and that CD8+ direct allorecognition stimulated by nonprofessional APCs plays a minor role.

Non-hematopoietic allograft cells directly activate CD8+ T cells and trigger acute rejection: an alternative mechanism of allorecognition.

Despite evidence that human non-hematopoietic cells, such as vascular endothelium, can activate allogeneic T lymphocytes in vitro, the prevailing view has been that hematopoietic antigen-presenting cells are required to trigger alloimmune responses in vivo. Here we report that mouse non-hematopoietic cells activate alloreactive CD8+ T lymphocytes in vitro and in vivo. We also show that vascularized cardiac allografts are acutely rejected via CD8+ direct allorecognition even if the alloantigen is not presented by hematopoietic professional antigen-presenting cells. Because activation of alloreactive CD8+ T cells by donor-type non-hematopoietic cells can continue for the life of the allograft, these findings present a new clinically relevant mechanism of allorecognition and should be taken into consideration when developing strategies to prevent allograft vasculopathy or to induce tolerance.

A novel small animal model of left ventricular tissue engineering.

BACKGROUND: Complex congenital cardiac anomalies involving ventricular hypoplasia require either staged palliative reconstruction, converting the circulatory system to a single ventricle based pump, or allogeneic transplantation. Tissue engineering offers the potential for complete reconstruction of these defects, but is limited by the inability to model myocardial tissue engineering in a small animal. Our goal was to develop a small animal model for ventricular tissue engineering using rat heterotopic heart transplantation. METHODS: Donor hearts were explanted after cardioplegic arrest and the left ventricular volume was augmented by the implantation of a biodegradable engineered construct. The heart was then transplanted heterotopically into syngeneic recipients creating either a volume loaded, functioning left ventricle, or a non-functioning left ventricle. Some of the engineered constructs were seeded with multipotent bone marrow-derived mesenchymal progenitor cells before implantation. Animals were evaluated by echocardiography, morphology, histology, and immunohistochemistry after 1 month. RESULTS: A scaffolding constructed from polytetrafluoroethylene, polylactide mesh, and type I and IV collagen hydrogel resulted in minimal intracardiac inflammation without aneurysmal dilatation. Successful transplantation and differentiation of mesenchymal progenitor cells was accomplished using this scaffolding. No ventricular arrhythmias resulted from this surgical manipulation and echocardiography revealed both end systolic and diastolic volume augmentation with ventricular expansion. CONCLUSION: We have developed an in vivo model of ventricular tissue engineering using heterotopic heart transplantation. Future work will focus on construction of ventricular tissue around pre-fabricated vascular networks in order increase cellular engraftment for ventricular reconstruction.