Family Repertoire Research Articles

VH gene family repertoires of “viable motheaten” (mev) mice

Mutant viable motheaten (mev) mice provide an useful experimental model to study the origin and molecular properties of autoantibodies. In the present investigation we have compared by in situ hybridization VH gene family usage in lipopolysaccharide-activated B cells (available repertoire) and spontaneously immunoglobulin-secreting (actual repertoire) B cells in the spleen of 6-8-week-old BALB/c and mutant BALB/c-mev mice. We have found that while sharing identical available splenic repertoires and expressing a diversified set of VH families, mev mice differ from control BALB/c animals in VH family representation in the actual plasma cell repertoires where they showed a decreased utilization of VH7183 genes and an increased representation of the VHJ606 family when compared to control BALB/c animals. These results indicate that selection of actual repertoires may indeed differ between autoimmune and control mice, but do not establish whether such changes are the primary cause of the disease or whether they are secondary to the initiating of the autoimmune process.

VH gene family repertoire of resting B cells. Preferential use of D-proximal families early in development may be due to distinct B cell subsets.

The fetal VH gene repertoire was shown previously to be characterized by overrepresentation of D-proximal families, VH 7183 and VH Q52, compared with adult bone marrow B cells in which VH genes were expressed in a more stochastic fashion. To determine the underlying mechanisms of these findings, adult vs fetal progenitors were placed in the same supportive microenvironment and the resulting B lineage cells analyzed for VH gene family expression. The supportive microenvironment was provided by established adult bone marrow stromal cell layers. In this way the relative importance of environmental vs genetic influences could be determined. The fetal B cells and pre-B cells that developed on adult stromal cells maintained a fetal-like VH gene family repertoire with preference for D-proximal families VH 7183 and Q52. In contrast, adult cultured B cells maintained the adult-like repertoire with predominance of the largest family VH J558. Only after long-term incubation was there a change in the expression of particular VH gene families. These findings suggest that the D-proximal VH gene family preference observed early in ontogeny is associated more with the inherent genetic potential of B cell progenitors that predominate during fetal life and less with environmental influences.

Comparison of V kappa gene family expression in adult and fetal B cells.

The functional B cell repertoires from adult and fetal mice were compared by examining V kappa gene family expression in individual cells. In addition, because little is known about the relative use of the various V kappa gene families in an immune response, adult B cells from several different strains of mice were analyzed. This was accomplished by stimulating B cells with the polyclonal activator, LPS. Activated cells were then analyzed for V kappa gene family expression at the single cell level by in situ hybridization using radiolabeled V kappa gene probes. It was found that all V kappa gene families tested were represented in the LPS-induced adult repertoire with V kappa 1, V kappa 4,5 and V kappa 19 being expressed to the largest degree in all strains tested. The LPS-induced adult V kappa gene family repertoire was then compared to the fetal repertoire and some differences were observed. In particular, a lower proportion of fetal B cells expressed V kappa 1 and a higher proportion of fetal B cells expressed V kappa 4,5 and V kappa 10. Importantly, compared with the adult response there was no evidence in the fetal response for an increased expression of V kappa 21, the family that maps closest to J kappa,C kappa. This is in contrast to what has been shown previously with H chain V region exons in which there was a clear preference for the VH gene families that mapped closest to DH.

Ig VH gene family repertoire of plasma cells derived from lupus-prone MRL/lpr and MRL/++ mice.

VH gene family usage was determined in both spontaneous, in vivo activated plasma cells and LPS-induced plasma cells from individual MRL/lpr mice by using in situ hybridization. It was found that VH gene family expression in spontaneous plasma cells varied from mouse to mouse. Some mice expressed VH families in an apparently random manner similar to that obtained with polyclonal activation. Other mice showed an exaggerated expression of particular VH gene families. VH J558 was overrepresented most frequently, but overrepresentation of VH 7183, Q52, and 36-60 was also observed. Importantly, LPS-induced VH gene family expression in these same mice displaying biased VH family usage in spontaneous plasma cells, appeared normal with no evidence for similar biases in the LPS-induced repertoire. Anti-DNA antibody concentrations and the degree of glomerulonephritis were determined for each mouse to measure the severity of disease. The level of expression of the J558 family was positively correlated with disease severity. The results suggest that the initial autoantibody response is highly diverse but becomes more restricted as the disease progresses.

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. II. IgG antibodies contain VH genes from a single VH family and VL genes from at least four VL families.

To define the V gene family repertoire of human IgG anti-Haemophilus influenzae type b polysaccharide antibodies, we purified six IgG1 and nine IgG2 anti-Hib-PS antibodies to monoclonality from immune serum of six individuals and performed N-terminal amino acid sequence analysis. Of the 15 clonal antibodies we examined, all H chain V regions were of the VHIII family. In contrast, the L chains of these antibodies were clearly from at least four different VL families; VKI, VKII, VKIII, and V lambda. Interestingly. VL family expression correlated with the cross-reactivity of these antibodies to the capsular carbohydrate of Escherichia coli K100. VKII antibodies did not cross-react, whereas antibodies expressing V lambda, VKI, or VKIII generally cross-reacted. We conclude that L chain V regions are very important contributors to the limited heterogeneity in this antibody repertoire.

Expression of antibody V-regions is genetically and developmentally controlled and modulated by the B lymphocyte environment

In this study we performed a comprehensive analysis of VH family usage in the emergent, available and actual repertoires of neonatal and adult BALB/c and C57BL/6 (B6) mouse strains. For this purpose we used an in situ hybridization technique that allows the detection of VH-gene expression at a single cell level. We have found that VH gene expression in neonatal mice is determined by a non-random position-dependent process which favours the utilization of the most D-proximal VH 7183 family. The preferential usage of the 7183 family is also characteristic of early differentiating bone marrow B cells of adult BALB/c mice. At different stages of ontogeny and B cell development VH family repertoires evolve in a strain-specific manner, with significantly higher utilization of the VH J558 family in B6 mice. In the peripheral immunocompetent cell pool, local environmental factors can further modulate VH family expression and lead to increased representation of the VH J558 family in peripheral lymph nodes and of the VH X-24 family in intestinal Peyer's patches. In conclusion, our present results indicate that VH family usage is controlled by genetic, developmental and environmental factors and suggest that selection of antibody repertoires can occur at multiple stages in B cell development.