Martine Lévy

In vitro generation of cells producing antierythrocyte autoantibodies in normal mice

Abstract The production of antierythrocyte autoantibodies was studied by a plaque assay. Plaque-forming cells (PFC) and hemagglutinating antibodies were produced upon in vitro culture of murine spleen cells in the presence of mouse red blood cells (MRBC). Kinetic experiments revealed that the plaque response reached a maximum after 5 to 6 days of culture. The generation of PFC required the presence of MRBC at the beginning of the culture. PFC numbers were highly increased when 1% horse serum was added to the culture medium. Horse serum was found to sensitize the plaque assay by interacting directly with MRBC. After filtration of horse serum on Sephadex G-200 columns, only the fraction eluting in the void volume increased PFC numbers and interacted with MRBC.

Molecular characterization of a monoclonal murine anti-blood group A antibody

A mouse monoclonal anti-human blood group A antigen (AC12, mu, kappa) has been generated and sequenced in order to analyze the immunoglobulin genes used to generate antibodies with anti-human blood group A specificity. Mice were immunized with human type A RBC. Anti-A producing hybridomas were detected by agglutination against human type A RBC. Total cellular RNA was extracted from hybridomas cells. PCR amplification and sequencing of anti-A heavy and light chain cDNAs were performed. The VH and VK sequences of antibody AC12 were shown to be very homologous to that used by other antibodies recognizing carbohydrates as well as glycoproteins, peptides or haptens constituting self antigens as well as nonself antigens. The VH sequence of antibody AC12 presented important homology with a previously reported monoclonal anti-blood group B antibody. The antibody AC12 also presented homology with the VH and VK sequences of a previously reported human anti-blood group A antibody which contributes additional evidence in favor of a restricted usage of V segments by antibodies directed against red blood antigens.

A phenotypic and functional analysis of long-lived B and T lymphocytes

The lymphocyte composition of spleen, lymph nodes, bone marrow, and thymus of mice submitted to hydroxyurea treatments for four consecutive days was studied. The treatment selects for small lymphocyte populations that represent between 4 and 20% of control numbers in the various organs. Spleen and bone marrow contain the same B cell population with a low IgM, high IgD, low I-E phenotype, which respond to LPS at control clonal frequencies. The T cell compartment is equally depleted, and the lymphocytes remaining contain frequencies of clonable cells in response to mitogens and IL-2 that are comparable to those detected in normal spleen cells. Overall, the results suggest that only a minor fraction of all lymphocytes in a normal young adult mouse have life spans longer than 4 days.

Sequence Homologies between a Monoclonal Murine Anti-Blood Group A and Antibodies Recognizing Carbohydrate Antigens of Red Blood Cells

A mouse IgM monoclonal anti-human blood group A (AC12) verified and approved by the National Reference Blood Group Center and currently used for ABO blood group typing has been sequenced. Very few data reporting the production and molecular characterization of monoclonal anti-A have been published so far [1]. The nucleotide heavy-chain sequence of antibody AC12 used a VH segment of the murine J606 family corresponding to the IGHV6 subgroup in the IMGT database. Interestingly, AC12 VH exhibited important homology (95.3%) with a monoclonal anti-blood group B (BC-1004) [1]. In addition, homology was also found with VH22.1, one of the germline genes of the J606 family specific for ß-(1,6)-galactan. As concerns CDR3, AC12 used the DSP2.2 segment (nucleotide 301 to 312) and the JH3 segment (nucleotide 313 to 345) in germline configuration.