In the present study we have compared by in situ hybridization and by a CFU-B colony assay VH family usage in the pre-B and B cell compartments of the bone marrow of adult BALB/c and C57BL/6 mice. We have found that the position dependent increased expression of the VH 7183 family, observed in neonatal mice, is characteristic of early differentiating B cells of adult mice. The quantitative analysis for each VH family of the number of pre-B and B cells produced daily and the number of mature B cells present in the peripheral immunocompetent cell pool of adult BALB/c demonstrates the existence of selecting mechanisms operating within the bone marrow at the level of the emergent repertoire and at the level of export of newly formed cells into the periphery. This selective process results in the decreased peripheral representation of the VH 7183 family and in the accumulation of cells belonging to the other VH families. Selection of VH family usage in peripheral repertoires may be determined according to lymphocyte life-span, as we have found a preferential utilization of the VH J558 family in populations of partially enriched, long-lived B cells.
We herein present a technical strategy to optimize DNA isolation from paraffin-embedded tissue (PET). This includes the choice of adequate buffers for proteinase K digestion and multiplex PCR amplifications for assessing the appropriateness of DNA extracts for subsequent PCR assays for detecting clonality. We found that the association of proteinase K digestion in nonionic buffer and subsequent extract dilutions accounted for 79% of successful amplifications. A final efficiency of 88% was achieved by additional organic extractions and/or re-extractions. Comparisons were carried out with control DNA extracts from fresh samples to assess the efficiency of each clonality assay. Immunoglobulin CDRIII rearranged region amplification was more efficient for pregerminal center B-cell lymphomas in contrast to CDRII rearrangement detection, which was more effective for germinal and postgerminal lymphomas. T-cell clonality detection by TCRgamma PCR was less efficient in PET samples than in fresh tissues showing that DNA integrity is more critical for TCR than for IGH amplification. Two inconclusive cases without phenotypic markers and two other atypical lymphoproliferations masked by reactive T cells were diagnosed as plasmablastic lymphomas and as monoclonal B-proliferations, respectively, due to IGH rearrangements.
Many adult splenic B cells die within 1 week in the spleen of adoptive adult recipient mice; in contrast, the cellular environment of newborn recipients allows for their expansion and persistence for several weeks. In the present study, we show that the local environment of adult peritoneal cavity also allows adult splenic B cells to persist for over 2 weeks after intraperitoneal transfer. In order to determine whether the persistence of donor B cells in newborn hosts and in the peritoneum of adult recipients results from a selection process involving the clonal specificities expressed, the variation in time of VH gene family repertoires of donor B cells was analyzed in the hosts. At different times after the transfer of splenic cells from lipopolysaccharide (LPS)-reactive mice into LPS-non responder histocompatible recipients, mRNA colony blot assays were performed. The results show that among the donor adult LPS-reactive B cells, the VH genes are differently used by the expanding or persisting B cells, in both kinds of recipients. Thus, cells expressing J558 or VH11 gene families are, in particular, positively selected, while those expressing D-proximal or J606 and 36-30 VH gene families are less selected. These findings demonstrate that the propensity of B cells to persist and expand is determined by their selection through their immunoglobulin variable regions, rather than by genetic properties linked to particular B cell subsets.
Lymphocyte populations in which Ly-1 B cells are differentially represented were studied for the expression of ten VH gene families, either by an RNA colony blot assay or by in situ hybridization of single cells, in BALB/c and C57BL/6 mice. The comparisons of cells from lymph nodes, Peyer's patches and adult spleen (poor in Ly-1 B cells) with cells from peritoneal cavity and neonatal spleen (rich in Ly-1 B cells) were confirmed by the analysis of adult peritoneal Ly-1- and Ly-1+ B cells sorted on the fluorescence-activated cell sorter. The results indicate that the peritoneal Ly-1+ B subset uses the whole spectrum of known VH gene families, and shows a preferential utilization of CP12 VH genes, most likely as a result of a selective process during life.
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