Antibody affinity maturation: Clonal selection at the level of the B lymphocyte surface.

Abstract

Affinity maturation of antibodies is achieved by antigen selection of specific B lymphocyte clones expressing membrane immunoglobulin (mIg). A model system was generated to examine antigen:mIg interactions by expressing mIg specific for a hapten, phosphocholine (PC). The murine B lymphoma M12.4 was transfected with various IgH constructs differently mutated in V$\sb{\rm H}$ and with an unmutated IgL construct, to produce stable B cell lines that differed in receptor density, affinity, and isotype. Increased receptor number correlated with increased calcium signaling at lower antigen concentrations for two series of mIgM$\sp+$ lines with low and 10-fold higher affinity for PC. Higher affinity similarly correlated with greater responses. Increased receptor number influenced antigen presenting cell (APC) function as shown by the production of more interleukin-2 by an ovalbumin-specific T hybridoma in co-culture experiments with B cell transfectants and PC-ovalbumin. B cells with higher affinity mIg were superior APC and were more efficient than lines with lower affinity mIg at higher receptor density when Ag was continuously present. Only lines with higher affinity mIg at higher density could demonstrate APC function in short-term Ag-pulse experiments; this was associated with greater Ag-induced capping of receptors. Similar results were obtained whether lines were single or double positive for mIgM and mIgD. Origin of the Ab response to the PC determinant of Proteus morganii (PC-PM) was explored, where a single somatic mutation in CDR2 of V$\sb{\rm H}$ confers significant Ag-binding ability. As opposed to the undetectable ELISA binding of Ab with unmutated CDR2, the mIg equivalent on B cell lines mediated PC-specific Ca$\sp{2+}$ flux upon PC-PM stimulation. Positive responses depended on high receptor number and were independent of isotypes $\mu$ and $\delta$ on single or double positive cells. Signaling was greater and more rapid in lines with higher affinity mIg (mutated in CDR2) even at low receptor density. Results were interpreted in the context of normal B cell development. Most mature B cells are $\mu\sp{low}$$\delta\sp{high}$. After Ag stimulation, receptor down-regulation is temporally related to V region somatic mutation. The parameters of mIg receptor number and affinity may work in concert to effect selection of B cell variants with higher affinity. A functional role for IgD is proposed, that of increasing the total number of receptors number to a high density to promote initiation of clonal selection.