Reconstitution of the B-cell repertoire following allogeneic bone marrow transplantation

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Immunology, Microbiology, Pathology and Infectious Diseases

Sammanfattning: Allogeneic bone marrow transplantation (BMT) is an established therapy for patients with a variety of haematological malignancies. Patients undergoing BMT often suffer from severe infections that can be partly explained by humoral immune deficiency. We have evaluated the molecular and serological basis for impaired humoral immunity among patients treated with BMT. Reconstitution of B-cell repertoires was followed from before to three years after transplantation by analysis of immunoglobulin (Ig) genes at DNA and RNA levels as well as by measurement of circulating antibodies in patients' sera. Four patients were analysed for Ig heavy chain variable (VH) gene usage. The diagnosis leading to transplantation was acute lymphoblastic leukaemia of pre-B type, lymphoma (K1+) and chronic myeloid leukaemia (two patients). Peripheral blood mononuclear cells were isolated from donated marrow and patients' venous blood. Nucleic acids were isolated and amplified with primers specific for various VH genes using PCR. Thereafter we analysed amplified fragments by nucleotide sequencing or hybridisation with oligonucleotide probes specific for individual B-cell clones. We detected a restricted usage of VH6 and VH3 Ig gene rearrangements among BMT patients compared to healthy controls. Complementarity determining regions (CDRs) 3 were characteristic of an adult type of rearrangement considering N-nucleotide additions and length. Furthermore, individual B-cell clones appeared to dominate the repertoire at different time points after transplantation. To evaluate the effect of the restricted VH gene usage on functional Ig repertoires, analysis of patient sera was performed. Sera from 44 patients, the majority receiving treatment for various haematological malignancies, were analysed with a quantitative immunoblot technique. This method is based on the Western-blot technique and allowed a global analysis of Ig specificities towards protein extracts prepared from human liver, muscle and skin tissue and from cultured Staphylococcus epidermidis. Quantification of reactive antibodies in each serum was conducted with densitometry. To analyse the immunoreactivity profiles and perform multivariate statistical treatment of the data dedicated computer software and macro routines were used. The results demonstrate that 60% of treated patients have severely reduced diversity in their IgM repertoire compared to healthy controls during and after the first year post-BMT. The IgG repertoire, however, is not affected and patients demonstrate a similar diversity of IgG antibodies as healthy controls. It is possible that the reduced diversity in the IgM repertoire, as well as the oligoclonal nature of VH6 and VH3 rearrangements among patients treated with BMT, contribute to the impaired humoral immunity characteristic of this patient group. The reduced diversity of the Ig repertoire might result in a decrease of the individual's capability to recognise and respond to certain bacterial or viral antigens. However, we demonstrate a more diverse IgM and IgG repertoire among BMT patients receiving marrow from a matched unrelated donor (MUD) than from a sibling donor. Patients receiving marrow from MUD suffer more infections than do patients receiving sibling marrow. From this it follows that a polyclonal repertoire might be secondary to immune activation by pathogens or minor antigen mismatches, and it might be that an oligoclonal repertoire, rather than contributing to sensitivity to infections, is a sign of lack of infections.

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