Sicco H. Popma

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.

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-γ-activated C57BL/6 (H-2b) vascular endothelial cells as stimulators and CD8+ T lymphocytes isolated from CBA/J (H-2k) mice as responders. Resting endothelium expressed low levels of MHC class I, which was markedly up-regulated after activation with IFN-γ. 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 CD44low naive CD8+ T lymphocytes. Activated CD8+ T lymphocytes had the ability to produce IFN-γ and IL-2, acquired an effector phenotype, and showed up-regulation of the antiapoptotic protein Bcl-xL. Treatment with CTLA4-Ig led to marked reduction of T cell proliferation and a decrease in expression of Bcl-xL. 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.

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.

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.

Replacement of graft-resident donor-type antigen presenting cells alters the tempo and pathogenesis of murine cardiac allograft rejection.

BACKGROUND Graft-resident antigen presenting cells (APCs) are potent stimulators of the alloresponse. To test whether replacement of graft-resident donor-type APCs with those of recipient-type alters allorecognition and the pathogenesis of both acute and chronic rejection, we created chimeric hearts for transplantation into naive recipients. METHODS To replace donor-type APCs with those of recipient-type, chimeric animals were created by bone marrow transplantation (BMT) in fully allogeneic mouse and rat strain combinations. The degree of APC replacement in chimeric organs was assessed phenotypically and functionally. Chimeric hearts were transplanted heterotopically into untreated recipients. RESULTS Flow cytometric and immunohistochemical analysis did not detect residual bone marrow recipient-type APCs in mouse BMT chimeras. Although semi-quantitative reverse transcription polymerase chain reaction detected 0.001-0.01% residual cells, APCs isolated from chimeric organs were functionally unable to stimulate donor-type cells. When transplanted into naive recipients, chimeric mouse hearts had significantly prolonged survival but were nevertheless rejected acutely. Similar results were obtained in the ACI --> LEW rat strain combination. However, in the PVG --> DA rat model, the majority of chimeric hearts survived >100 days and all long-surviving hearts developed cardiac allograft vasculopathy. CONCLUSIONS BMT leads to near complete replacement of organ-resident APCs. The virtual absence of donor-type APCs in chimeric hearts delays or prevents acute rejection in a strain-dependent manner. In contrast, this type of graft modification does not prevent cardiac allograft vasculopathy. This suggests that, although the CD4+ direct pathway may play a role in acute rejection, it is not essential for the development of chronic rejection in rodent cardiac allografts.

A simple method for culturing mouse vascular endothelium.

Vascular endothelium is an important site for a wide array of immunological processes such as inflammation, atherosclerosis and allograft rejection. Culture methods of mouse vascular endothelium would provide an important in vitro correlate to immunological murine in vivo models. We describe a simple method to culture mouse vascular endothelium from thoracic aorta. Our cultured cells express typical phenotypic (CD105, CD31, CD106), morphological and ultrastructural (intercellular junctions, Weibel-Palade bodies) markers of vascular endothelium. They also possess functional receptors for uptake and processing of acetylated low-density lipoproteins. The mouse vascular endothelium within our system expresses high levels of MHC class I and MHC class II after activation with IFN-gamma. In addition, these cells express the accessory molecules CD80 and CD54, while they lack constitutive expression of CD86 and CD40, providing them the means to function as antigen presenting cells. Alloreactive CD4(+) and CD8(+) T lymphocytes demonstrate evidence of DNA synthesis after co-culture with activated vascular endothelium indicating their commitment to proliferation. In conclusion, we describe a simple culture system to isolate and grow mouse vascular endothelium, which provides a powerful tool to study biological interactions in vitro.

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.

Immune monitoring in xenotransplantation: The multiparameter flow cytometric mixed lymphocyte culture assay

Xenotransplantation requires monitoring of complex cellular interactions in vitro. A tool to monitor cell proliferation in detail would be instrumental in understanding these cellular interactions in heterogeneous xenogeneic lymphocyte cultures and in patients after xenotransplantation. To accomplish this, we used a fluorescent cell proliferation marker, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE), in combination with flow cytometry. CFSE, a green fluorescent molecule, binds covalently to intracellular macromolecules. Each cell division reduces the fluorescent intensity per cell by half and shows a characteristic multipeak pattern in flow cytometric analysis. For this study, human lymphocytes were labeled with CFSE and cultured in the presence of irradiated porcine lymphocytes. Cell proliferation was detected in CFSE-labeled lymphocytes in both a single and a multiparameter flow cytometry setting. Concurrently, tritiated ((3)H) thymidine incorporation, a common method to measure gross cell proliferation, was assessed. The kinetics of CFSE-labeled cell proliferation correlated with (3)H-thymidine incorporation in that both methods showed a lag phase for days 1-3 and a log phase for days 4-7. Multiparameter flow cytometric monitoring of mixed lymphocyte cultures allowed phenotyping and assessment of viability of proliferating populations in heterogeneous xenogeneic stimulated human lymphocyte cultures and complemented the classical (3)H-thymidine incorporation assay. The use of this technique will allow a wide array of immunologic parameters to be measured in a heterogeneous xenogeneic mixed lymphocyte culture. The information gained from these assays is essential to understanding the biological significance of xenogeneic cellular interaction and for monitoring the immune status of the xenotransplanted patient.

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.

Flow Cytometric Test for Beryllium Sensitivity

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.