Alternative mechanisms of receptor editing in autoreactive B cells

Abstract

Pathogenic anti-DNA antibodies expressed in systemic lupus erythematosis bind DNA mainly through electrostatic interactions between the positively charged Arg residues of the antibody complementarity determining region (CDR) and the negatively charged phosphate groups of DNA. The importance of Arg in CDR3 for DNA binding has been shown in mice with transgenes coding for anti-DNA V(H) regions; there is also a close correlation between arginines in CDR3 of antibodies and DNA binding. Codons for Arg can readily be formed by V(D)J rearrangement; thereby, antibodies that bind DNA are part of the preimmune repertoire. Anti-DNAs in healthy mice are regulated by receptor editing, a mechanism that replaces κ light (L) chains compatible with DNA binding with κ L chains that harbor aspartic residues. This negatively charged amino acid is thought to neutralize Arg sites in the V(H). Editing by replacement is allowed at the κ locus, because the rearranged VJ is nested between unrearranged Vs and Js. However, neither λ nor heavy (H) chain loci are organized so as to allow such second rearrangements. In this study, we analyze regulation of anti-DNA H chains in mice that lack the κ locus, κ-/κ- mice. These mice show that the endogenous preimmune repertoire does indeed include a high frequency of antibodies with Arg in their CDR3s (putative anti-DNAs) and they are associated mainly with the editor L chain λx. The editing mechanisms in the case of λ-expressing B cells include L chain allelic inclusion and V(H) replacement.

Fig. 1.

L chain isotype use in B6 wild-type (Left), B6 κ-/κ- (Center), and 56R B6 κ-/κ- (Right) mice. Splenocytes were stained with PE/Cy7 anti-B220, Alexa-647, anti-λX specific monoclonal antibodies (kindly provided by P.A. Cazenave (57)] and FITC-anti-λ1,2,3-monoclonal antibodies for B6 wild-type and B6 κ-/κ- and PE anti-λ1 antibody (L22.18.2, kindly provided by U. Storb) for 56R B6 κ-/κ- plots. The dot plots display λ1,2,3 for Left and Central panels and λ1 only for the Right panel (y axis) vs. λX expression (x axis) on cells in the live, lymphoid, B220+ gate.

Fig. 2.

V_H gene family use in the splenic B cells expressing editor (λX, dark bars) and noneditor (λ1,2,3, light bars) L chains from B6 κ-/κ- mice as determined by single cell reverse transcription-PCR. V_H use is depicted as the relative frequency of each V_H gene family among all analyzed V_H sequences within each sorted population. N = 215 (100%) for λX and N = 88 (100%) for λ1,2,3. V_H genes that were found only in λX antibodies are indicated with asterisk (*).

Fig. 3.

V_H family Sm7 gene use and V_H CDR3 amino acid sequences of H chains in B cells sorted on the basis of L chain expression and expressed with editor (VλX), noneditor (Vλ1,2,3), or both (Vλ1,2,3/VλX) L chains from B6 κ-/κ- mice. Args in each CDR3 are highlighted in red.

Fig. 4.

V_H replacement footprints. (A) Schematic representation of V_H replacement. White triangles represent RS sequences. Dark triangles represent the cRSS or embedded heptamer. The green region represents a rearranging V_H (donor) that replaced the initially rearranged V_H (recipient, magenta) and joined to the short stretch of nucleotides (footprint, magenta) that remained after the recipient V_H was cleaved at cRSS. (B) Potential V_H replacement products were found among H chain sequences from splenic B cells from κ-/κ- mice. Donor V_H refers to the V_H gene segment fully represented in the complete V(D)J sequence. The nucleotide sequences shown are the end of the V_H donor sequence and the N1 nucleotides located between the last nucleotide of framework 3 and the first D nucleotide. Potential V_H replacement footprints are defined as 5 or more consecutive nucleotides of identity with the 3′ end sequence of a nondonor germline V_H and are highlighted in magenta. Possible recipients are germline V_Hs whose 3′ end sequence contains the footprint sequence found in the V_H replacement product. The amino acids shown are from the V_H CDR3. P nucleotides are in parentheses.