Cellular and Molecular Aspects of the Germinal Centre Reaction

Sammanfattning: The humoral immune response to a T cell dependent protein antigen will over time generate antibodies with an increased affinity to the antigen. This is due to a process known as affinity maturation and proceeds in a, for this purpose, well designed micro-milieu within secondary lymphoid organs known as germinal centres. The increase in affinity of an antibody in, e.g. mouse and man is due mainly to incorporation of single base-pair mutations, so called somatic mutations, in combination with an efficient competitive re-selection of better binders on the antigen held in its naïve form on follicular dendritic cells. Studies on affinity-maturation in human B cells and in cells within the germinal centre, have been hampered by the fact that the cells of interest die rapidly in vitro and the lack of a proper cell culture system for study of such cells. The aim of this thesis is to design such a cell culture system that allows growth of single human germinal centre B cells, known to accumulate somatic mutations in vivo, to study these single cell cultures for in vitro mutational activity, and to investigate signals required to induce naïve B cells towards a germinal centre B cell phenotype in vitro. By the use of a rapid screening system for in vitro accumulation of somatic mutations (RT-PCR and SSCP), clones raised from single cells were analysed for in vitro mutational activity. Human tonsillar, in vivo primed, germinal centre B cells were cultured together with murine EL-4 cells and shown to preserve some of their most characteristic extracellular phenotype and proliferation capacity. Still, no further in vitro accumulation of mutations were found in such single cell culture clones. Though, combination with interleukin-supplemented cultures based on CD40 crosslinking (via CD32 transfected murine L cells) of human GC-B cells revealed that their mutational activity could be preserved with one of the interleukin-combinations used, pointing out the importance of soluble factors for preserving the germinal centre reaction as well. In addition, signals required in vitro to differentiate resting B cells to a germinal centre B cell phenotype were identified. It was shown that stimulation via sIg and CD40, in combination with CD44, seem to drive the B-cell towards a GC-B cell phenotype. The role for CD4+CD57+ GC-T cells were also examined, and were shown to be able to rescue GC-B cells in vitro and drive peripheral blood B cells to Ig production by the addition of exogenous IL-2.