Stochastic pairing of heavy-chain and kappa light-chain variable gene families occurs in polyclonally activated B cells.

Abstract

Frequencies of 25 immunoglobulin heavychain and K light-chain variable (VH + VK) gene-family pairings expressed in splenic B-cell populations were determined by hybridization of VH-and Va-family-specific DNA probes to mitogen-induced B-cell colonies from C57BL/6 mice or hybridomas derived from BALB/c and NZB mice. Both analyses support the conclusion that VH and VK gene families pair without bias; as would be expected for random association, the frequencies of specific VH + V.K pairs may be estimated by the product of the independent VH and VK frequencies. Based upon the frequencies at which 9 VH and 12 VK gene families are expressed, we calculated the expected usage for -100 VH + VK family pairings in neonatal and adult C57BL/6 mice. Variability in the expression of such VH + VK pairings is considerable; pairs representing >10% to <0.01% of the splenic B-cell population occur. This variability is most pronounced in the neonate, where 6 VH + VK family pairs account for nearly 40% of all mitogen-reactive B cells. As the neonate matures, the distribution of frequencies for VH + VK pairings becomes more nearly uniform. This process may underlie the patterned acquisition of humoral immune responsiveness.We and others have investigated the frequencies at which families of immunoglobulin heavy-chain and K light-chain variable (VH and VK) genes are expressed in mice during development (1-7). In general, the frequencies at which VH gene families are productively rearranged in adult mice is stoichiometric, that is, proportional to VH family size (1-3). However, in fetal or neonatal mice VH expression is biased for those VH exons located in the 3' region of the locus (2). In contrast, neither is the expression of VK gene families in the adult mouse stoichiometric nor is there bias for the use of 3' exons in the neonate (5-7).Here we address the combinatorics of specific VH and VK family pairings by determining the frequencies at which several VH and VK families are expressed both independently and in combination. Two experimental strategies were employed. First, the filter paper disc method for lymphocyte cloning was used to screen large numbers of lipopolysaccharide (LPS)-induced B-cell colonies for VH and VK expression (8). Sequential hybridizations of single discs permitted identification of C57BL/6-derived B-cell clones expressing speCifiC VH + VK family pairings (9). Second, two panels of hybridomas, derived from LPS-activated BALB/c or NZB splenocytes, were created by selection of clones positive for expression of the X-24 VH family (BALB/c) or expression of the VK1 gene family (NZB). Subsequently, all clones in each panel were retyped for either VK or VH expression. The results ofboth experimental strategies support the conclusion that VH and VK families pair without bias. Thus, if some VH family X is expressed among B cells at frequency x, and a VK 10029; and tDepartment of Microbiology and Immunology, University of family Y at frequency y, the measured frequency of B cells expressing both (X + Y) families...

Ig Allotypic Inclusion Does Not Prevent B Cell Development or Response

“…Nonetheless, the presence of dual B cells in the MZ B cell subset was supported by the fact that the frequencies of dual events in the B cell population (Fig. 1B) and of dual MZ figure), anti-human L chain F(abЈ) 2 (␣hIg in figure), 20 g/ml LPS, 15 g/ml anti-CD40 plus 50 ng/ml IL-4, or a combination of anti-IgM and anti-CD40 plus IL-4 for 16 -18 h and analyzed for hC and mC, as well as CD69 and CD86 expression by flow cytometry. The geometric mean of fluorescence intensity for both CD69 and CD86 in single human (hSP, Ⅺ), dual (DP, u), and single mouse (mSP, f) B cells are shown in this figure, which represents one of three independent experiments.…”

“…Briefly, 96-well Nunc-Immuno plates (Nalge Nunc International) were coated with 2 g/ml goat F(abЈ) 2 antihuman -chain polyclonal Ab (Southern Biotechnology Associates) in PBS containing 150 mM NaCl and 0.1% NaNa 3 . Plates were washed once with PBS, 0.5% Tween 20, and blocked with PBS, 30% FBS, and 0.05% NaNa 3 at 4°C overnight.…”

“…L chain isotypic inclusion (i.e., the expression of both a L chain and a L chain on a single B cell) and allelic inclusion (i.e., the expression of two distinct V H or V L from two alleles of the same locus) result in cells with the potential to express two different H ϩ L chain combinations and, therefore, specificities. There is strong evidence to indicate that Ͼ90% of genotypic allelic inclusions translate in phenotypic inclusions (expression of both H ϩ L chain combinations), whereas Ͻ10% manifest in phenotypic exclusions (expression of only one H ϩ L chain combination) probably as a result of poor H ϩ L chain pairing (2,3). One of the proposed reasons for the evolution of haplotype exclusion in B cells is that this process ensures negative selection of autoreactive cells.…”

T15-Idiotype-Negative B Cells Dominate the Phosphocholine Binding Cells in the Preimmune Repertoire of T15i Knockin Mice

“…Therefore, among the PC-binding cells, usage of these specific V genes is 1.5-to 4.1-fold greater than that observed in the total B cell population. However, virtually all of the PC-dex binding colonies, irrespective of the V gene used, were rearranged to J 5, whereas, in LPS-activated B cells, 20% of the colonies express J 5 (53,54). This means that the V 19 -15:J 5, V 8 -28:J 5, and V 22-33:J 5 genes are in fact expressed at a 7-, 13-, and 21-fold higher frequency in PC-binding cells than expected in the total B cell population.…”

“…Of the total B cells present in T15i mice, PC-specific cells expressing V 22-33, V 8 -28, or V 19 -15 genes represent 0.74, 2.5, and 1.4%, respectively. In LPS-stimulated B cells from normal mice, the frequency of expression of these V genes is calculated to be 0.18, 0.95, and 0.96%, respectively (53,54). Therefore, among the PC-binding cells, usage of these specific V genes is 1.5-to 4.1-fold greater than that observed in the total B cell population.…”