Cellular and molecular events involved in the initiation of the immune response to Proteus morganii.

Abstract

Previous work done in our lab has shown that secondary immune responses to the phosphocholine epitope of Proceus morganii (PC(PM)) are dominated by the progeny of a few progenitor cells, and that these antibodies have numerous somatic point mutations. These mutations are found most frequently in the heavy chain and include numerous examples of mutations which recur in independent immune responses. Because the secondary response is so restricted, we wanted to understand how this immune response is initiated. The first portion of this thesis characterizes the onset of the response to PC(PM). The principle observation discussed in Chapter One is that antibodies derived from the primary immune response have the same pattern of mutations seen in the secondary immune response. Moreover, antibodies lacking all but one of these mutations do not appear to be reactive with PC(PM). This observation results in a biological paradox. If precursor antibodies are not reactive with antigen, how can an antigen specific immune response be initiated? Next, we wanted to determine the molecular basis of a recurring mutations characteristic of the anti-PC(PM) specificity that is seen in 100% of all anti-PC(PM) antibodies. A cDNA library representing the pre-immune repertoire was screened with an oligonucleotide probe specific for the recurring mutation. Six clones were identified which contained this mutation. Sequence analysis of these clones suggests that this mutation is a result of V to DJ joining. This result allows us to define the molecular precursor of the anti-PC(PM) immune response. Finally, we developed an experimental approach based on generating Ag specific transfectomas. The B-cell lymphoma, M12.4 was transfected with a series of heavy chain constructs which contained combinations of recurring mutations which are known to effect binding specificity. Assay of these cell lines for their ability to mobilize calcium in response to antigen indicates that somatic point mutations are necessary to create specificity to PC(PM). A model for the initiation of the anti-PC(PM) immune response is that within a certain window of time, a cross-reactive antigen must stimulate somatic hypermutation in the anti-PC(PM) precursor cell resulting in a PC(PM) reactive immune response.