▼ BACKGROUND: Dendritic cells (DCs), regulators of adaptive and innate immunity, are also major players in adoptive immunotherapy. DCs are professional antigen presenting cells that can be generated in vitro from different precursor cells and using different culture environments. There are several different DC generation procedures described in the literature; each generate functional DCs, but with variable surface antigen expression and cytokine production. As a result, the activated T-cells, and thus, the immune response is determined by the phenotypic and functional properties of the DCs. Therefore, it is imperative to choose precursor cells and a culture environment that best suit the intended therapeutic application. STUDY DESIGN AND METHODS: This study aimed to generate monocyte-derived dendritic cells, to induce their maturation using two different cytokine combinations, and to compare the two combinations for their effects on DCs phenotypic and functional properties. Peripheral blood mononuclear cells (PBMCs) were separated from peripheral blood by density gradient centrifugation. After monocyte enrichment by plastic adhesion, PBMCs were cultured for five days in the presence of granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) to generate the immature DCs. Immature DCs were then matured by culture for 48 hours in the presence of one of two cytokine combinations (CC). CC-1 includes GM-CSF, IL-4, tumor necrosis factor (TNF-α), and prostaglandin E-1 (PGE-1). CC-2 includes, IL-1β, IL-6, TNF-α, and PGE-2. Immature or mature DCs were assayed for the expression of surface antigens CD83, CD86, and CD209, for the production of cytokines IL-10, IL-12p40, and IL-12p70, and for the uptake of fluorescein isothiocyanate (FITC) conjugated Dextran. RESULTS: On average, 1 mL of peripheral blood was sufficient to generate 2x105 CD209+ immature DCs. CC-1 matured DCs showed a significantly higher CD83 expression, and higher IL-12p40 production after maturation, but no IL-10 was detected. CC-2 matured DCs, however, showed IL-12p40 production, and significant IL-10 production, but moderate CD83 expression. Both combinations resulted in decreased ability of DCs to uptake Dextran, and high CD86 and CD209 expression. Further, both combinations failed to induce production of detectable IL-12p70.CONCLUSION: Immature monocyte-derived dendritic cells respond differently to different cytokine combinations, resulting in phenotypically and functionally distinct DC subsets. The resulting phenotypic and functional variability between CC-1- and CC-2-matured dendritic cells is expected to result in activation of two different subsets of T cells. Advisors/Committee Members: Leemhuis, Thomas.

▼ Background: Neutrophil antibodies compromise the innate immune response and can also initiate transfusion related acute lung injury, the number one cause of transfusion related mortality. Neutrophil antibody and antigen detection is technically challenging; few clinical laboratories perform such testing. Our objective was to establish procedures to detect human neutrophil antigens (HNA) and their corresponding antibodies. Methods: Neutrophils were molecularly typed for HNA-1a, and -1b using sequence specific primers (GTI Diagnostics, Waukesha,WI). Results were used to establish two serologic tests, a granulocyte indirect immunofluorescence test (GIFT) and a granulocyte agglutination test (GAT), capable of detecting HNA-1a, 1b, 2a, and 3a. Based on strength of HNA expression in the serologic assays, a neutrophil panel was developed and used to test sera previously screened for anti-HNA by a reference laboratory. Results: GAT reported an Accuracy probability of 97% for HNA-1a and -1b phenotyping and a Positive Predictive value of 100% and 96%, respectively. GIFT reported the HNA-1a and -1b phenotype with an accuracy probability of 97% and 100%, respectively and exhibited 97% and 100% Positive Predictive value. As an antibody screening assay, GIFT demonstrated an average Negative Predictive value of 78% and an average Sensitivity of 81%. Conclusion: This study significantly advanced neutrophil serology at Hoxworth Blood Center. The phenotyping assays demonstrated excellent correlation with the genotypes reported by GranType™ and the antibody screening assay demonstrated good correlation with the results reported by the Blood Center of Wisconsin. Future studies involving antibody identification, through murine monoclonal antibody inhibition or conventional methods should be explored. Also, better elucidation of the antibody screening positive cut-off should be explored in order to maximize the probability of the assay finding no anti-HNA among those who do not have anti-HNA. Increasing the Specificity of anti-HNA screening will enhance the clinical application of the neutrophil antibody screening assay, an important tool in the accurate identification of alloantibody-induced TRALI. Advisors/Committee Members: Susskind, Dr. Brian.

▼ BACKGROUND: Transfusion-Related Acute Lung Injury (TRALI), one of the most common noninfectious, life-threatening implications of blood transfusion,is associated with the administration of blood that contains anti-MHC (Major Histocompatibility Complex) antibodies. To date, theories regarding TRALI pathogenesis are based on in vitro animal studies and human clinical case studies. Lack of controlled in vivo animal experiments has made it difficult to test theories of TRALI pathogenesis as well as possible therapeutic interventions. Our objective was to develop an in vivo murine model that shares features with human TRALI. METHODS: BALB/c (H-2d) mice were injected with 34-1-2s, a murine IgG2a anti-H-2d (D/K) monoclonal antibody (mAb). Hypothermia which reflects the development of shock) was measured by rectal thermography over the next 2 hours and dyspnea was measured by noninvasive barometric plethysmography. RESULTS: Hypothermia and dyspnea were rapidly induced in BALB/c mice but not in congenic H-2k controls. Pathologic changes in the lungs after anti-MHC mAb challenge included an increase in vascular permeability that led to pulmonary edema and protein leak into the alveolar spaces. In addition, a neutrophilic alveolar infiltrate developed within two hours of challenge. This evolved into a mononuclear alveolar infiltrate by 24 hours after challenge. Pretreatment with anti-FcγRII/III mAb to block FcγRII/III receptors, gadolinium to deplete macrophages, anti-granulocyte mAb to deplete granulocytes, or platelet activating factor (PAF), histamine or leukotriene antagonists, inhibited the induction of hypothermia and dyspnea. CONCLUSIONS: These results suggest that IgG, FcγRIII, granulocytes, macrophages, PAF, histamine, and leukotrienes are important in anti-MHC antibody induction of shock and pulmonary dysfunction. This animal model should be useful for determining the pathogenic mechanisms of TRALI and for identifying potential therapeutic interventions. Advisors/Committee Members: Susskind, Dr. Brian.

▼ Blood cell formation is a highly regulated process that depends on the proliferation and differentiation of hematopoietic stem cells (HSC). HSC are located in the endosteal area of the bone marrow (BM) cavity and their function depends on an intimate relation with osteoblasts and stromal (O/S) cells. The factors involved in this intercellular interaction are not known. We proposed that direct cell-to-cell intercellular communication (IC) at the endosteum is crucial for normal hematopoiesis, specifically under stress conditions. The best known form of IC is through channels called gap junctions (GJ), which allow the passage of small, charged molecules, including secondary messengers that may trigger intracellular signaling responses. GJ are formed by hexamers of proteins called connexins (Cx). One of these, connexin-43 (Cx43), is ubiquitously expressed and is the Cx most prevalent in BM. Cx43 is normally expressed at low levels in BM but administration of 5-fluorouracil (5-FU), a chemotherapy drug, has been shown to increase Cx43-expression in BM approximately 100-fold. Using an inducible, conditional Cx43 knock-out murine model, we found that Cx43 is critical for adult hematopoiesis after 5-FU administration. Here we analyzed the microlocalization of BM Cx43-expression after 5-FU administration, and confirmed that under stress conditions, Cx43-expression is predominantly located in the endosteum. Next, the hematopoietic function of Cx43-deficient mice was analyzed. We observed that Cx43-deficient mice displayed a major impairment in hematological recovery after 5-FU administration, which included myelo-, erythro- and thrombopoiesis. This correlated with a significant depletion of the cellularity and the myelo-erythroid progenitor content of the BM and spleen in Cx43-deficient mice. This depletion did not affect the HSC population, which concomitantly became enriched in BM. Competitive repopulation of Cx43-deficient HSC showed impaired short-term, but not long-term repopulation when compared to wild-type (WT) counterparts. Although a proliferation defect of Cx43-deficient mice cannot be ruled out, we found no significant decrease in the proliferation status of BM and spleen HSC in vivo in Cx43-deficient mice on Day 9 after 5-FU administration. Since Cx43 is expressed by O/S and HSC, we analyzed the effect of Cx43-deficiency in full hematopoietic chimeras, where either the O/S or the HSC were deficient in Cx43. The results suggest that HSC from WT Cx43 mice transplanted into mice with a Cx43-deficient expression in O/S cells induces significant monocytopenia and lymphopenia (pandlt0.05) in peripheral blood. Further, chimeric mice that express Cx43 in O/S cells and transplanted with Cx43-deficient HSC displayed leukopenia with neutropenia, monocytopenia and lymphocytopenia. This suggests that Cx43-expression plays a crucial role in both the HSC and O/S cell components. In summary, Cx43-expression in the BM endosteum appears to play a major role in the hematopoietic response to stress conditions. Advisors/Committee Members: Cancelas, Dr. Jose A.

▼ BACKGROUND: Donor leukocyte infusions (DLI) have recently become more widely used as therapy for the treatment of disease relapse after allogeneic stem cell transplantation. DLIs have been shown to mediate the crucial graft vs. tumor effect needed to reach remission. The white blood cells that make up a DLI, specifically T cell populations, will recognize and destroy cancer cells and will facilitate engraftment by combating residual host T cell populations.¹ The ability to store these therapeutic cells until they are needed is important, but there is inadequate data regarding the analysis of their function after cryopreservation. Routine cryopreservation methods currently consist of protocols designed for stem cell storage, however it may be important to apply different techniques to different cell types, such as T cells. It is vital to the nature of this therapy that T cells remain viable and functional post thaw. STUDY DESIGN AND METHODS: This study was conducted in order to compare two widely used cryopreservation processes, currently used for hematopoietic stem cells, and determine their ability to retain T cell viability and function. Peripheral blood mononuclear cells (PBMCs) from normal donors were cryopreserved in 10% dimethylsulfoxide (DMSO) or 5% DMSO with 3% hetastarch (HES). Fresh vs. frozen/thawed samples were then compared post thaw to determine T cell viability and recovery. To evaluate T cell proliferation, cell cycling ability, and cytokine production, specifically IFN-γ, T cells were mitogen stimulated and evaluated at different time points in culture. RESULTS: No significant differences were found between cryopreservation methods in the post thaw viability and recovery of T cells. In addition, cell cycle distribution was not significantly different between fresh and frozen cells. However, there was a clear delay in the ability of the cells cryopreserved with 10% DMSO to produce IFN-γ. This 10% DMSO method also showed a delay in its ability to support T cell expansion. CONCLUSIONS: The cells frozen in 5% DMSO and 3% HES did not behave significantly different than fresh cells in terms of cell expansion and IFN-γ production. T cell products cryopreserved for DLI in 10% DMSO showed a disadvantage, in vitro, in their ability to proliferate and produce IFN-γ in response to PHA stimulation. Advisors/Committee Members: Leemhuis, Dr. Thomas B.

▼ Cell-to-cell contact between hematopoietic stem cells and progenitors (HSC/P) and their supporting bone marrow (BM) microenvironment has been shown to be pivotal in blood formation and hematopoietic homeostasis between BM and peripheral blood. BM derived endothelial cells (EC) form a major cell component in the hematopoietic microenvironment. BMEC are fenestrated to allow the traffic of hematopoietic cells to and from the circulation while maintaining adhesion and communication with other EC and HSC/P. Gap junctions (GJ) represent one system in which adjacent cells adhere in order to communicate intercellularly. GJ are channels constituted by a group of proteins called connexins (Cx). It is known that Cx43 GJ are involved in the interactions between hematopoietic cells and the BM microenvironment. Based on in vivo preliminary data indicating loss of retention of HSC/P in BM of endothelial-specific Cx43-deficient mice, we hypothesized that the loss of Cx43 would induce a decreased adhesion and/or increased migration of HSC/P, respectively, to or through Cx43-deficient EC.Our results indicate that the deficiency of Cx43 in EC, induced by RNA interference induces decreased adhesion and increased transendothelial migration of normal HSC/P. However, the transendothelial migration of Cx43-deficient HSC/P through Cx43 RNA-silenced EC is not modified, suggesting that the mechanisms mediated by Cx43 involved in the control of transendothelial migration are more complex than anticipated. In summary, Cx43 deficiency in the EC compartment may induce a complex array of changes in the proliferation, adhesion and migration of HSC/P. Migration of HSC/P, however, seems to depend on the expression of Cx43 in both EC and HSC/P. Cx43 plays pleiotropic, cell-specific roles in the hematopoietic microenvironment. Advisors/Committee Members: Cancelas-Perez, Jose.

▼ Adult bone marrow mesenchymal stem cells and progenitors (BM MSC/P) constitute an attractive source of potentially transplantable cells for therapy of skeletal diseases. A murine model of BM MSC/P might be helpful in the design of approaches to correct specific genetic and skeletal diseases by gene delivery after MSC/P transplantation. Feasibility of isolation, ex vivo expansion, transduction and transplantation of BM MSC/P in a murine experimental model were investigated. For isolation and ex vivo expansion, BM MSC/P isolation based on plastic adherence and culture under defined conditions followed by macrophage depletion (method A) was compared with a hematopoietic/endothelial-depleted (CD45-/TER119-/CD11b-/CD34-/CD31-) bone marrow population (method B). Multilineage differentiation of MSC/P into osteoblasts, chondrocytes and adipocytes was also assayed in tissue-specific, medium-defined conditions. Transduction of MSC/P was performed with the retroviral MIEG3 vector expressing Enhanced Green Fluorescent Protein (EGFP). For analysis of skeletal tissue engraftment, 1) whole BM cells from smooth muscle alpha-actin/EYFP (SMAA8-EYFP) or smooth muscle gamma-actin/EGFP (SMGA13.7-EGFP) transgenic mice, or 2) ex vivo expanded MSC/P expressing either an alpha-actin-driven EYFP or EGFP driven by a constitutively active retrovirus promoter (MIEG3-transduced) were transplanted. A total of either 10e6 BM cells or 2 x 10e4 MSC/P were injected intravenously via the tail vein of 7 Gy-irradiated syngeneic recipients. Method A yielded higher colony-forming-units of fibroblast type (CFU-F) content. MSC/P isolated and expanded by method A were easily transduced using retroviral vectors and grew exponentially until reaching greater than 10e14-fold and 471-fold cell and CFU-F expansion, respectively. Exponentially growing MSC/P could be differentiated into adipocytes, osteoblasts and chondrocytes and we observed that transplantation of SMAA8-EYFP but not SMGA13.7-EGFP transgenic BM cells resulted in patches of spindled cells surrounding pre- or post-sinusoidal vessels in the BM of non-transgenic recipient mice. The feasibility of the expansion and transduction of MSC/P in a murine model and BM stroma transplantation by systemic infusion was demonstrated. These data confirm that MSC/P comply with the major requirements of a cell vector for gene therapy: efficient propagation and transduction ex vivo, together with the ability to be genetically loaded and transplanted. Advisors/Committee Members: Cancelas, Dr. Jose A.