Journal: The Journal of Experimental Medicine

Article Title: Follicular lymphoma-like B cells in healthy individuals: a novel intermediate step in early lymphomagenesis

doi: 10.1084/jem.20061292

Figure Lengend Snippet: t(14;18) + B cells in the peripheral blood of HI are not naive B cells and constitute an expanding population of FL-like clones. (A) Human PBMCs from 10 consecutive blood samples were collected from anonymous healthy donors, and IgD + /CD27 − naive, IgD + /CD27 + memory, and IgD − /CD27 + switched memory B cell populations were isolated by cell sorting. Results from a representative cell-sorting experiment are shown. (B) Genomic DNA from the three purified subsets was tested for the presence of t(14;18) using the short-range BCL2/J H PCR assay (mbr20A/21A and J H coB/cointB nested primers; see ). A two-step “fluctuation” nested PCR assay was used, consisting of at least five parallel replicates performed for each sample (lanes 1–5). Results from sample #20 using 100 ng per replicate is depicted. (C) The contribution of naive (CD27 − , white circles) and memory (CD27 + , black triangles) B cells to the total t(14;18) + frequency (CD19 + , gray squares) in the healthy donors is shown. The translocation frequency in each subset was plotted for each donor . The 10 samples are ordered according to increasing CD19 + total B cell translocation frequencies (gray squares). (D) PCR fragments from CD27 + memory B cells were cloned and sequenced, and clonality was identified on the basis of the BCL2/J H junction, which is unique for a given translocation. The frequency of each independent BCL2/J H clone was calculated, and the contribution of each clone (vertical bars, each color represents a given clone in a given sample) was superimposed on the t(14;18) frequency from the CD27 + subset (black triangles). All positive amplicons were sequenced except one in sample #12, three in #15, and two in #24 (percentage on top of histograms corresponds to the number of sequenced amplicons on the total number of positive replicates; ). For #99, which required DNA dilution for the calculation of the frequency, 100% corresponds to the sequencing of 10 out of 10 clones obtained from amplifications performed on diluted DNA (1:5; ) and undiluted DNA (9:9; not depicted). (E) BCL2-Sγ (top) and BCL2/Sμ LR-PCR (bottom) were performed in the 3 IgD + /CD27 − naive, IgD + /CD27 + memory, and IgD − /CD27 + switched memory B cell subsets for four HI (#15, #20, #21, and #24). Results from sample #24 are shown. β-globin/TCR-β controls were performed as control for long-range amplifications (unpublished data). Most positive amplicons were confirmed by cloning/sequencing and alignment to germline BCL2 , Sμ, and Sγ loci (not depicted). Samples presenting multiple size bands revealed oligo/polyclonality. (F) The overall distribution of switched (gray histograms) versus unswitched (white histograms) translocated alleles in the three B cell subsets from samples #15, #20, #21, and #24 is represented. The ratio of positive replicates is indicated at the top of the bars.

Article Snippet: The following antibodies were used: IgD biotinylated, CD19-APC (BD PharMingen), CD27-PE, CD20-FITC (Immunotech), and IgD-FITC (Caltag Laboratories).

Techniques: Clone Assay, Isolation, FACS, Purification, Nested PCR, Translocation Assay, Sequencing, Long Range PCR, Control, Cloning

Journal: The Journal of Experimental Medicine

Article Title: Follicular lymphoma-like B cells in healthy individuals: a novel intermediate step in early lymphomagenesis

doi: 10.1084/jem.20061292

Figure Lengend Snippet: Analysis of BCL2/J H translocations in total PBMCs and FACS-sorted B cell subsets from healthy donors

Article Snippet: The following antibodies were used: IgD biotinylated, CD19-APC (BD PharMingen), CD27-PE, CD20-FITC (Immunotech), and IgD-FITC (Caltag Laboratories).

Techniques: