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. 2017 Jun 21;8:15869. doi: 10.1038/ncomms15869

Figure 3. Somatic mutations occur in expanded CD8+ T-cell clones that persist during follow-up.

Figure 3

(a) The figure shows the CD8+ clonal architecture of patient 1, on the basis of TCRB sequencing and V-J genes. The Vβ13.1+ population detected by flow cytometry corresponded to clones using TCRBV06-05, 06-06, or 06-09 genes. (b) TCRB sequencing showed that patient 1 harboured two very large CD8+ T-cell clones. The two expanded clones of patient 1 persisted at a similar level during follow-up. The amino-acid TCRB sequences and the V genes of these unique clones are shown in the figure. (c) Amplicon sequencing of FACS-sorted cell fractions confirmed the identified mutations. The table presents the VAFs in each cell fraction. (d) The clonal architecture of patient 2's CD8+ pool as shown via V and J genes. Clones using TCRBV09-01 correspond to Vβ1+ antibody in flow cytometry. (e) Similarly presented TCRB sequencing results of flow-sorted Vβ1+ cells. When examining unique clones (‘unique’ defined by a unique nucleotide sequence) the largest clone composed 73% of all CD8+ Vβ1+ cells. (f) Patient 2 harboured several unique CD8+ T-cell clones at diagnosis. The TCRBV09-01 clone, in which mutations were observed, increased slightly during the follow-up. Amino-acid sequences derived from these unique clones are shown. The clone using TCRBV09-01 in this panel had the exact same nucleotide TCRB sequence as the largest clone in sorted Vβ1+ cells. (g) Amplicon sequencing results on FACS-sorted cells show the VAFs in each cell fraction. The low (<1%) VAFs found in CD4+ and Vβ13.6+ cells are considered sorting impurities, and thus the mutations in patient 2 occur exclusively in CD8+Vβ1+ cells. The mutation VAFs in CD8+Vβ1+ cells correspond well with the TCRBV09-01 clone size in sorted cells (h) The clonal landscape of CD8+ cells from patient 3. Clones using TCRBV04-03 gene correspond to Vβ7.2 usage in flow cytometry. (i) Patient 3 harboured a very large CD8+ T-cell clone at diagnosis, which persisted at a similar level during follow-up. (j) Amplicon sequencing of flow-sorted cell populations showed that the mutations detected in exome sequencing exist only in Vβ7.2 CD8+ T cells and not in other T cells.