A somatic second-site mutation in the T-cell compartment is associated with oligoclonal expansion of T cells. (A) Quantitative sequence analysis of the CARD11 gene from the indicated cell populations. Percentages indicate the frequency of the mutated allele (Mutation Surveyor DNA Variant Analysis software). (B) Alterations of the CARD11 sequence and its consequences on protein level are shown. (C) TCRVβ usage among CD8⁺ T cells in the bone marrow as assessed by flow cytometry using TCRVβ chain-specific antibodies. The percentage of CD8⁺ T cells expressing the indicated TCRVβ chain is shown for P1 compared with reference values.²⁸  (D) The indicated T-cell populations were purified from bone marrow of P1 by FACS sorting (>95% purity) for quantitative sequence analysis as in (A). (E) TCRγ rearrangement analysis of CD4⁺, CD8⁺/Vβ20⁺, and CD8⁺/Vβ20^– T cells isolated from bone marrow of P1 using primers for Vγ1-8 (blue) and Vγ10 (black). Amplicon length (in bp) is plotted against the fluorescence intensity (arbitrary units). Size standards are indicated in orange. (F) Vβ20-specific primers were used to amplify the Vβ region of FACS-sorted CD8⁺/Vβ20⁺ T cells (left) and PBMC from a healthy control (right). (G) PCR products from (F) were sequenced and V(D)J junction sequences were analyzed with IMGT/V-Quest (imgt.org).