T.25. Elucidating the Pathogenicity of Mutations in the C2 Domain of Perforin in Hemophagocytic Lymphohistiocytosis

S58 were devoid of CD21 expression. Western blot analysis revealed complete absence of CD21 protein expression in the patient. A compound heterozygous mutation in the CD21 gene was identified as the underlying molecular defect, leading to premature mRNA decay, respectively to a rapidly degraded CD21 protein. Despite reduced class-switched memory B cell numbers the patient mounts protective and long-lived specific antibody responses. This is in contrast to the severely impaired capacity of patient's B cells to bind C3d-containing immune complexes. Further experiments detecting alterations in CD21 dependent calcium flux in the patient are in progress. Our findings also impact the biology of EBV infection and associated human disorder. We provide evidence for EBV infection of human B cells in vitro and in vivo in the absence of CD21 expression, and we could demonstrate almost absent binding of EBV gp350 to the patient's B cells. doi:10.1016/j.clim.2009.03.164

T.24. Allogenic Stem Cell Transplantation in Three Patients with Common Variable Immunodeficiency Carla Neumann, Sigune Goldacker, Reinhard Marks, Marta Rizzi, Klaus Warnatz, Martin Werner, Juergen Finke, Hans Hartmut Peter. University Medical Center Freiburg, Freiburg, Germany Common variable immunodeficiency (CVID) is the most common primary immunodeficiency (PID). CVID is characterized by reduced antibody responses to immunisation and infectious pathogens. Therefore the patients need regular immunoglobulin substitution. CVID patients have an increased risk of lymphoma. While other PID were successfully treated with allogenic stem cell transplantation (SCT) to date, SCT has not been published as treatment option in CVID patients. We report here three cases of SCT in adult male CVID patients with long standing disease. The diagnosis of CVID was made at the ages of 27, 28 and 38 years, respectively. Two patients were transplanted because of a secondary lymphoma (T-LGL, angioimmunoblastic lymphoma), the third patient because of progressive hypersplenism and pancytopenia. All three SCTs were performed with HLA identical related donors after a conditioning regimen of fludarabin, carmustin and melphalan. Engraftment was successful in all three patients but one patient died three months after the procedure because of generalized adenovirus infection and delayed myeloid regeneration. The other two patients stayed in remission regarding their lymphoma, but remained hypogammaglobulinemic and still require immunoglobulin substitution therapy two years post transplant. The susceptibility to infections was unchanged and the markedly reduced class-switched B cell memory had not recovered. These case reports demonstrate that allogenic SCT is a treatment option for lymphoma in CVID while it did not cure the underlying immunodeficiency. CVID patients may require special pre-and post transplant care in order to avoid unusual infectious complications. doi:10.1016/j.clim.2009.03.165

Abstracts

T.25. Elucidating the Pathogenicity of Mutations in the C2 Domain of Perforin in Hemophagocytic Lymphohistiocytosis Ramon Urrea1, Juana Gil2, Carmen Rodríguez-Sáinz2, Elena Cela3, Lisa Filipovich1, Andrew Herr4, Janos Sumegi1, Michael Jordan1, Kimberly Risma1. 1Cincinnati Childrens Hospital, Cincinnati, OH; 2Hospital Gregorio Marañon, Madrid, Spain; 3Hospital Gregorio Marañon, Madrid, Spain; 4 University of Cincinnati College of Medicine, Cincinnati, OH Familial hemophagocytic lymphohistiocytosis type 2 (FHL-2) is a severe inflammatory disorder associated with mutations in perforin, a critical molecule for lymphocytemediated pathogen elimination and immune homeostasis. A number of FHL-2 associated mutations are located in the C2 domain of perforin, thought to be responsible for calciumdependent, lipid-binding to target cell membranes. Recently we identified two C2 mutations in a family affected by FHL: T435M, a previously described mutant with disputed pathogenicity and Y438C, a novel substitution. Interestingly, those family members with T435M/Y438C genotype were healthy and exhibited normal cytotoxic function. To predict their risk of FHL, we utilized a multi-approach analysis to address structure and function, both by expressing the recombinant proteins in perforin deficient cells and by biophysical modeling. Both mutant perforins were secreted upon stimulation as revealed by novel antibody-capture and liposome-binding assays. We found that T435M abolished perforin cytotoxicity by inhibiting its calcium-dependent lipid binding, while, perforin-Y438C retained function, despite the complete absence of proteolytic maturation. We have subsequently tested additional C2 mutations to confirm our molecular model. Finally, experiments are ongoing to elucidate whether the precursor form of WT perforin is a truly functional form, thus challenging the historical model that perforin maturation is a pre-requisite for perforin function. doi:10.1016/j.clim.2009.03.166

T.26. Readjusting the T Cell Receptor Signal Threshold- A New Therapeutic Target in Acute Coronary Syndrome Sergey Pryshchep, Jorg Goronzy, Cornelia Weyand. Emory University, Atlanta, GA Background: Clinical complications of atherosclerosis, in particular acute coronary syndromes (ACS), result from T cell and macrophage-mediated tissue damage in the atherosclerotic plaque. The current study was designed to explore whether T cell receptor (TCR) activation thresholds in ACS patients are lowered leading to illegitimate activation and plaque-damaging T effector functions. Methods: CD4 T cells were isolated from the blood of 80 patients admitted with ACS and 77 age/ sex-matched controls. TCR responsiveness was assayed with a stable source of superantigen-loaded antigen-presenting cells (APC). TCR signaling events were quantified by confocal microscopy and ELISA. Results: CD4 T cells from ACS patients stimulated by superantigen-loaded APC proliferated and