Daniel Kreisel

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.

BACKGROUNDnComplex 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.nnnMETHODSnDonor 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.nnnRESULTSnA 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.nnnCONCLUSIONnWe 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.

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.

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.

Strain-Specific Variation in Murine Natural Killer Gene Complex Contributes to Differences in Immunosurveillance for Urethane-Induced Lung Cancer

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide and results from a complex interaction between carcinogen exposure and inherent susceptibility. Despite its prevalence, genetic factors that predispose to the development of lung cancer remain elusive. Inbred mouse models offer a unique and clinically relevant tool to study genetic factors that contribute to lung carcinogenesis due to the development of tumors that resemble human adenocarcinoma and broad strain-specific variation in cancer incidence after carcinogen administration. Here, we set out to investigate whether strain-specific variability in tumor immunosurveillance contributes to differences in lung cancer. Using bone marrow transplantation, we determined that hematopoietic cells from lung cancer-resistant mice could significantly impede the development of cancer in a susceptible strain. Furthermore, we show that this is not due to differences in tumor-promoting inflammatory changes or variability in immunosurveillance by the adaptive immune system but results from strain-specific differences in natural killer (NK) cell cytotoxicity. Using a newly discovered congenic strain of mice, we show a previously unrecognized role for strain-specific polymorphisms in the natural killer gene complex (NKC) in immunosurveillance for carcinogen-induced lung cancer. Because polymorphisms in the NKC are highly prevalent in man, our data may explain why certain individuals without obvious risk factors develop lung cancer whereas others remain resistant to the disease despite heavy environmental carcinogen exposure.

Calcium ionophore activation of chronic myelogenous leukemia progenitor cells into dendritic cells is mediated by calcineurin phosphatase

We have previously demonstrated that Ph+ myeloid progenitor cells of patients with chronic myeloid leukemia (CML) can acquire characteristics of mature dendritic cells (DC) following calcium mobilization with calcium ionophore (A23187, CI). In this study we characterize the intracellular signaling pathway by which CI induces the acquisition of DC features in these leukemic cells. CI-induced activation of CML cells is attenuated by the calcineurin phosphatase inhibitor cyclosporin A (CsA) as well as the calmodulin (CaM) antagonist W-7. These cause ablation of both the CI-induced immunophenotypic expression of DC markers and immunostimulatory properties in mixed leukocyte responses (MLR). Minimal blocking effect was observed when Ca(2+)/CaM kinase II (281-301) inhibitor was added to the cultures. These findings suggest a Ca(2+)-dependent mechanism for the CI-induced activation of CML cells into antigen-presenting cells (APC), which is primarily mediated through the CaM/calcineurin pathway.

Myocardial tissue engineering and regeneration as a therapeutic alternative to transplantation.

Ischemic cardiomyopathy leading to congestive heart failure remains the leading source of morbidity and mortality in Western society and medical management of this condition offers only palliative treatment. While allogeneic heart transplantation can both extend and improve the quality of life for patients with end-stage heart failure, this therapeutic option is limited by donor organ shortage. Even after successful transplantation, chronic cardiac rejection in the form of cardiac allograft vasculopathy can severely limit the lifespan of the transplanted organ. Current experimental efforts focus on cellular cardiomyoplasty, myocardial tissue engineering, and myocardial regeneration as alternative approaches to whole organ transplantation. Such strategies may offer novel forms of therapy to patients with end-stage heart failure within the near future.

Emergent lung retransplantation after discovery of two primary malignancies in the donor.

A 50-year-old woman underwent single lung transplantation for advanced chronic obstructive pulmonary disease. Shortly after the procedure, it was discovered that the donor suffered from both a renal cell carcinoma and a spindle-cell sarcoma of the ascending aorta, which had metastasized to the spleen. The patient was emergently listed for a retransplantation and underwent bilateral lung transplantation after a new donor became available 4 days after the initial transplantation procedure. After 24 months, the patient is without evidence of malignancy. This case illustrates the role of immediate retransplantation for patients who have inadvertently received thoracic organs from donors harboring occult malignancies.

Interleukin‐2‐mediated Survival and Proliferative Signals are Uncoupled in T Lymphocytes that Fail to Divide after Activation1

T lymphocytes are heterogeneous with respect to their ability to proliferate following activation in vitro and in vivo. Approximately 30% of T lymphocytes fail to progress through the cell cycle, despite showing evidence of an activated state. The population of T lymphocytes that remains undivided during a primary stimulation has been shown to be refractory to restimulation via the TCR and fails to proliferate in response to IL‐2. In an in vitro model of T‐cell deletion following clonal expansion, we demonstrate that T lymphocytes that do not progress through the cell cycle during primary stimulation have a sevenfold greater survival advantage compared with T lymphocytes that have divided. Progression through multiple division cycles is associated with down‐regulation of Bcl‐2 during a postactivation period of growth factor withdrawal. However this alone does not account for diminished survival, as constitutive expression of a Bcl‐2 transgene did not restore survival to the levels seen in undivided cells. Engagement of the IL‐2 receptor on these undivided activated T lymphocytes leads to enhanced survival and up‐regulation of Bcl‐2 and Bcl‐xL. Surprisingly, while IL‐2 also induces phosphorylation of Akt, it does not initiate cell cycle progression in this population of primary undivided cells. Our data provide evidence that a T cells survival capacity is linked to its proliferative behavior. Furthermore, our results provide the first report of a population of T cells, in which the IL‐2 receptor‐mediated signaling pathways leading to survival and proliferation are naturally uncoupled.

Die Rolle des Genotypes der organständigen passageren Leukozyten bei der immunologischen Akzeptanz der Rattenleber

Hintergrund: Rattenlebern werden bei bestimmten MHC-inkompatiblen Stammkombinationen spontan akzeptiert. Es ist vorgeschlagen worden, das dieses Pha nomen auf zellula rem Mikrochima rismus im Empfa ngertier beruht, ein Status, welcher durch passagere Leukozyten im Spenderorgan induziert wird. Wir untersuchten, ob passagere Leukozyten vom Spender-Genotyp eine Voraussetzung fu r die immunologische Akzeptanz der Rattenleber im Transplantationsmodell sind.