ALBERT

Description: Proteinase 3 (P3), a serine protease found in primary granules in granulocytes, is the target of T cell- and B cell-mediated autoimmunity in Wegener’s granulomatosis (WG) and of anti-leukemia immunity mediated by PR1 (VLQELNVTV)-specific cytotoxic T lymphocytes (PR1-CTL). Although aberrant P3 and neutrophil elasase (NE) expression in leukemia increases susceptibility to PR1-CTL-mediated killing, overexpression of P3 also induces apoptosis of the high affinity PR1-CTL leading to deletional tolerance and leukemia outgrowth. Because expression of P3 and NE in sera from leukemia patients is increased by 5-fold compared to healthy controls, we sought to determine whether such overexpression of P3 or NE impairs PR1-CTL immunity to leukemia by a direct affect on T lymphocytes. To study this, T cells from healthy donors were activated by anti-CD3 and anti-CD28 and exposed to increasing concentration of P3 or NE over one to five days, and the percentage of apoptotic cells and cell proliferation were determined by flow cytometry using PI, anti-Ki-67, and CFSE. P3, but not NE, induced dose-dependent apoptosis of up to 30% of T cells, and in the non-apoptotic cells, a 50% inhibition of CD4 and CD8 T cell proliferation at 1 μg/ml and 100% at 10 μg/ml compared to untreated cells. This effect was not enzyme-mediated since prior exposure of P3 to 56°C or co-incubation with the serine protease inhibitors Elafin and alpha-1 antitrypsin showed no affect on apoptosis or cell proliferation. P3 induced a cell cycle arrest at the G0/G1 interface, determined with PI and Ki-67 staining of healthy donor T cells that were exposed to P3 for up to 3 days. In contrast, at protein concentrations up to 25 μg/ml, NE showed no such inhibitory effect on apoptosis or cell proliferation. In addition to its role as a leukemia-associated antigen, P3 is also targeted by the cANCA antibody in patients with WG and the serum titer correlates with disease activity. Therefore, we hypothesized that the effect of P3 on T cell proliferation might also be affected by humoral immunity during circumstances of systemic autoimmunity. Co-incubation of P3 with a molar excess of cANCA reversed P3- mediated inhibition of both CD4 and CD8 T cells, consistent with a role of this antibody and the P3 target antigen in controlling T cell autoreactivity. Taken together, this data shows a new role for P3 in regulating T cell proliferation, which occurs only at high P3 concentration, similar to P3 in sera from leukemia patients, which is not enzymedependent. This supports a direct role for P3 in regulating both anti-leukemia immunity and autoimmunity. This data will need to be considered for effective immunotherapy targeting P3 in leukemia patients and these inhibitory effects also suggest a role for P3 in regulating autoimmunity at sites of inflammation, such as in patients with WG.

Description: Background: Microscopic peripheral blood and bone marrow cell evaluation remains a milestone in the diagnostic of hematology. Many factors contribute to the lack of standardization of this diagnostic test, such as differences in the procedure, staining, degree of skill in interpretation and glossary. However the new WHO classification highlights the importance of morphological aspects, quantitative as well qualitative, for a better stratification of patients in the diagnosis of haematological malignancies, particularly myeloid malignancies and above all myelodysplasia (MDS). In this high technology era, we have the opportunity to exchange, via the internet, images and information without geographic limitation, sparing time and resources. This represents a great opportunity to try to get a consensus for blood cell morphology. The European LeukemiaNet (ELN) Network of excellence is an EU project funded by the 6th FP, involving 174 centres from 28 countries. Its major goal is the construction of a cooperative network for improving leukemia diagnosis, care and research. Within the activities of the Diagnostic Platform (WP10), focused on Flow Cytometric and Morphological panels, a European Morphology Consensus Faculty (EMCF), composed of 21 expert morphologists from 13 European countries, has been organized with the following goals: to harmonize the identification of hematological cells for a common European morphological diagnostic pathway, in terms of cell lineage, maturation level, normality/abnormality and glossary, to take into account specific national skills, competences and methods, to provide the patients with the same morphological diagnosis all over Europe. The first step was to create a consensus-based cell library of meaningful blood cell images identified and named by top level European morphologists and agreed by EMCF, as a valuable tool of traininig. We are presenting the final results of the first study of the EMCF. Methods: From May to July 2007 we collected from all the EMCF members 164 images containing 438 labelled blood cells with the following distribution: Granulocytic series n=126 (28.5%), Erythroid series n=77 (17.5%), Monocytic series n=35 (8%), Lymphoid series n=107 (24.,5%), Megakaryocytic series n=23 (5.,5%), Blasts (not otherwise specified) n=29 (6.5%) Other n= 41 (9.5%). All the images were uploaded to a restricted web page together with an Excel file containing the author’s proposal for each cell. Faculty Members were asked to fill the Excel file with their agreed or personal alternative definition for each labelled cell. On January 2008 cells lacking a full agreement were submitted to a Delphi scoring procedure: a minimum of 3 agreements was needed for a cell to be scored in the Delphi questionnaire. By July 2008 we have received the Delphi files for the final evaluation of the study results. Evaluation rules: Full consensus: cells with an agreement of at least 17 out of 21 (〉80%) EMFC members. Delphi questionnaire for all the cells with an agreement 59% of all submitted cells. The main discrepancies in the morphological consensus concern the groups of blast and monocytic series, while for the other groups of cells the distribution is similar. After the Delphi scoring we reached an agreement on all the 216 submitted cells. The EMCF created a new category “Cell to delete” for 8 cells due to a not perfect quality of images. This image library is composed of 430 cells now identified through a consensus method by a European Morphology Consensus Faculty and the cells will be uploaded onto the ELN web site, with a clear identification of those cells identified with a Delphi scoring procedure. Moreover cell names are harmonized and this represents the first morphological European glossary. Future developments: The second extended Faculty including morphologists from 16 European countries has been formed with the aim to apply the consensus glossary to identify a new set of cells submitted by the authors without any suggestions. A meeting is planned to discuss the results.