B-cell repertoire reconstitution after bone-marrow transplantation evaluated by Ig heavy chain third complementarity determining regions (H CDR3s) fingerprinting: experience in 14 pediatric patients.

D. Di Martino PhD 1, P. Di Michele PhD 1, L. Scarso PhD 2, P. Terranova PhD1, M. Miano MD 1, S. Dallorso MD 1, E.Lanino MD 1, G. Dini MD 1, A.Valetto PhD 3

1 Department of Pediatric Hematology and Oncology; 2 Transfusional Service, Giannina Gaslini Institute, Genoa, Italy; 3 Department of Molecular Genetics, A.O. S.Chiara, Pisa, Italy .

Immunomediated complications are the most important problem after bone marrow transplantation (BMT). In particular Graft-versus-host-disease (GVHD) and infections represent frequent causes of morbidity and mortality; thus, a analysis of immunological reconstitution of the B-cell repertoire can be useful to monitor and modulate post-transplantation therapies. 14 pediatric patients referred to our Institute for BM transplantation were enrolled in this study. Samples were obtained before and after transplantation at various timepoints (+15, +30, +60, +90, +180 and +365) and evaluated by CDR3 fingerprinting. Briefly, this technique allows to get information about the diversification of the B-cell repertoire, analyzing the length of the H CDR3, one of the hypervariable segments of the IgH that provides essential residues for direct interaction with antigens. For CDR3 analysis, RNAs were isolated from mononuclear cells; cDNA was synthesized and amplified by two consecutive polymerase chain reaction (PCR): in the first were used an Ig-family-specific primer (VH3 or VH6) in combination with a primer for the constant region (IgM); the nested PCR was performed with forward and a reverse primers located in the framework 3 and JH region of the Ig gene, respectively. PCR products were then separated on 6.5% denaturing polyacrylamide gel and silver stained. The typical pattern obtained by CDR3 fingerprinting in a normal healthy donor is characterized by about 16-20 bands. Each band corresponds to a particular length of the CDR3, whereas in monoclonal B-cell diseases only one strong band is detected. Patients analyzed just after transplantation show a strong oligoclonality (few CDR3 bands detected, as expected) to return gradually to a normal situation (> 10 bands detected) around 3-6 months after transplantation. The molecular data were also correlated with data obtained by fluorescent activated cell sorter (FACS) analysis with a large panel of Monoclonal Antibody (MoAbs) to analyze B-cell antigen expression and coexpression. Our data can represent a preliminary work to understand the kinetic of B-cell immuno-repertoire reconstitution in children, showing that immune deficiency appears to be due to functional and numerical B-cell defects as it has been described in adult patients.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Stem Cell Biology.