A model for interclonal competition in the germinal center: Dynamic selection processes by-pass the affinity dead-end of low affinity anti-NP specific B cells

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Doctoral Thesis
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Acs, Andreas

The GC reaction is a highly competitive process in which high affinity B cells are favored to survive and proliferate in order to generate Abs of high affinity. To further analyze this competition, focusing on the fate of low affinity B cells, we made use of a transgenic transfer system. The mice used for this model express a transgenic heavy chain which, when paired with a λ1 LC, generate NP-specific Abs of low affinity. When splenocytes of such mice were transferred into wt recipients, B1-8lo GC B cells are reproducibly outcompeted between day 6 and 9 of the anti-NP response. This competitive phase was accompanied by an accumulation of B1-8lo GC B cells in the LZ together with a decreased frequency of proliferative B1-8lo cells in the DZ. We were further able to shed light on the molecular mechanisms leading to the disadvantage of B1-8lo GC B cells. A W33L mutation which is canonically found to be selected in C57BL/6 mice in response to NP is known to enhance affinity 10-fold. As expected, affinity maturation of endogenous GC cells led to a positive selection of cells bearing this mutation. In contrast, B1-8lo GC B cells did not show any selection of W33L, resulting in a selective disadvantage due to the absence of an increased affinity. Through generating recombinant Abs and measuring their affinity to NP, we were able to show that one of the four pre-existing amino acid exchanges within the B1-8lo heavy chain sequence was sufficient to reduce the affinity of an unmutated B1-8 Ab 10-fold. Hence, this amino acid change solely explains the low affinity of the B1-8lo Ab. Furthermore, the same amino acid exchange was found to interfere with an affinity enhancing effect of W33L, preventing B1-8lo B cells to stay competitive, being outcompeted by endogenous cells. To shed light on the fate of B1-8lo GC B cells during competition, we analyzed apoptosis and memory B cell (MBC) differentiation. Although the frequency of apoptotic B1-8lo GC B cells was increased compared to endogenous cells, the majority of these cells did not bind NP. It remains unclear whether this is due to increased apoptosis of B1-8lo GC B cells that exhibit another specificity than NP or due to the fact that surface molecules are downregulated on apoptotic cells, which might lead to impaired Ag binding. Surprisingly, we found B1-8lo cells to largely contribute to CD38hiFas-NP+ B cells, which are supposed to mainly contain MBCs. Interestingly, the frequency of this population remained fairly constant at all time points analyzed, although NP-specific B1-8lo B cells were concurrently outcompeted in the GC. Although it needs to be proven that this population indeed contains MBCs, a contribution of low affinity B cells to the MBC pool has been described before. Nevertheless, the extent of this contribution might give new insights into the fate of low affinity B cells which are unable to compete in the GC reaction.

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