Counterselection against D mu is mediated through immunoglobulin (Ig)alpha-Igbeta

Abstract

The pre-B cell receptor is a key checkpoint regulator in developing B cells. Early events that are controlled by the pre-B cell receptor include positive selection for cells express membrane immunoglobulin heavy chains and negative selection against cells expressing truncated immunoglobulins that lack a complete variable region (D mu). Positive selection is known to be mediated by membrane immunoglobulin heavy chains through Ig alpha-Ig beta, whereas the mechanism for counterselection against D mu has not been determined. We have examined the role of the Ig alpha-Ig beta signal transducers in counterselection against D mu using mice that lack Ig beta. We found that D mu expression is not selected against in developing B cells in Ig beta mutant mice. Thus, the molecular mechanism for counterselection against D mu in pre-B cells resembles positive selection in that it requires interaction between mD mu and Ig alpha-Ig beta.

Figure 1

Flow cytometric analysis of spleen (Spl) and bone marrow cells (BM) from Igβ^−/−, transgenic, and wild-type mice. Single-cell suspensions from lymphoid organs of 6-wkold mice were prepared and analyzed on FACScan^®. Bone marrow cells were stained with PE–anti-B220 and FITC–anti-CD43. Spleen cells were stained with FITC–anti-IgM and PE– anti-B220. The lymphocyte population was gated according to standard forward- and side-scatter values. The numbers in each quadrant represent the percentages of gated lymphocytes. WT, wild type; RAG ^−/−, RAG-1 mutant; Igβ ^−/−, Igβ mutant; Igβ ^−/− μ.TG, Igβ mutant, mIgμ, transgenic.

Figure 2

Ig gene rearrangements in Igβ^−/−, transgenic, and wild-type mice. Bone marrow DNA from 6-wk-old mice was amplified with VH558L, VH7183 (not shown), or D_H and J_H2 primers. Control primers were from the J-CH1 intervening sequence (IVS) (22).

Figure 3

Nucleotide sequences of D_H–J_H joints from Igβ^−/− and wild-type sorted bone marrow B cells. D_H segment, N or P nucleotides, and J_H4 segment sequences are shown. The number of DJ_H4 joints in D_H RF 1, 2, or 3 are noted. Stop codons in D_H segments are underlined.

Figure 4

Nucleotide sequences of V(D)J (VH558L to DJH4) joints from Igβ^−/− and wild-type bone marrow B cells. D_H segment, N or P nucleotides, and J_H4 segment sequences are shown. The number of DJ_H4 joints in D_H RF 1, 2, or 3 are noted. Stop codons in D_H segments are underlined, and the D_H segments used (S) are indicated.