Conventional and Surrogate Light Chains Differentially Regulate Ig μ and Dμ Heavy Chain Maturation and Surface Expression

Abstract

Positive selection of precursor (pre-) B cells by Ig membrane μ H chains (μm HC) and counterselection mediated by the truncated HC Dμ depend on the ability of each HC to form a pre-B cell receptor (pre-BCR) signaling complex with the surrogate L chain (SLC) components λ5 and Vpre-B. To better understand how pre-BCR signaling output is determined by its Ig components and the SLC, we investigated the regulation of pre-BCR surface expression and HC secretory maturation in a new nonlymphoid system. We took this approach as a means to distinguish B-lineage-specific effects from pre-BCR-intrinsic properties that may influence these aspects of pre-BCR homeostasis necessary for signaling. As in pre-B cells, the SLC in nonlymphoid cells supported only a limited degree of μm HC maturation and low pre-BCR surface expression levels compared with conventional LCs, indicating that this was due to an intrinsic property of the SLC. We identified the non-Ig region of λ5 as harboring the restrictive activity responsible for this phenotype. This property of λ5 was also evident with Dμ, but the overall SLC- and L chain-dependent requirements for Dμ maturation and surface expression were markedly different from those for μm. Surprisingly, Dμ was modified in an unusual manner that was only dependent on Vpre-B. These results establish a novel function of λ5 in limiting surface pre-BCR levels and reveal biochemical properties of Ig molecules that may underlie the diverse consequences of pre-BCR signaling in vivo by different HCs.

Multiple Levels of Selection Responsive to Immunoglobulin Light Chain and Heavy Chain Structures Impede the Development of Dμ-Expressing B Cells

“…The creation of cDNAs encoding the mouse H chain 17.2.25, D, the human H chain TG.SA (T), 1, MOPC, and JC have been described previously (16). Briefly, JC was made by replacing the leader and V sequence of a MOPC21 L chain with the leader of 1.…”

“…HEK293 cells were cotransfected by calcium phosphate-mediated precipitation of MiG-L chain (4 g), pEBB-H chain (4 g), pEBB-Ig␤ (2 g), and pEBB-Ig␣ (2 g) plasmids as described (16). Empty pEBB vector was used to normalize the total amount of DNA introduced to cells.…”

“…Two days later, cells were harvested by incubation with PBS supplemented with 10 mM EDTA, followed by pipetting into single-cell suspensions. Aliquots were prepared for flow cytometry and Western blot analysis as described (16).…”

“…One objective was to establish the relationship of the ability of a L chain to fold autonomously and be secreted to pairing with D. This is because in our studies and in those of others (15,16), the representative 1 L chain was secretion competent, whereas the L chains were not and depended on association with a full-length H chain for ER release and surface expression. It was hypothesized that this may be an important parameter limiting L chain/D association because D lacks a V H region with which a V L could fold and pair.…”

“…A molecular correlate to the signaling impairment is that D-preBCRs fail to be transported out of the endoplasmic reticulum (ER) to reach post-ER compartments and the cell surface as efficiently as normal preBCRs with full-length H chains (14 -18). Mutational analysis of the SLC demonstrated this was in part because VpreB requires a V H partner for this to occur optimally (16).…”