Fig 1.
Flow cytometric staining of TCRVβ subunits reveals clonal expansions in ATL patients.
Cryopreserved PBMCs from 52 individuals (28 ATL; 11 AC; 13 HAM) were thawed and stained with a viability stain followed by antibodies specific for 24 TCRVβ subunits, CD3, CD4, CD8, CADM1, CD7, CD127, CD25 and CCR4. Proviral genomic integration sites were mapped by LM-PCR and HTS. OCI was calculated using the Gini index, as previously described [12]. (A) Representative data from one individual with chronic ATL, and one high PVL AC. Pie charts show the relative frequency distribution of unique integration sites (green), and CD3+ cells (TCRVβ identified: CD4+, red; CD8+, blue; TCRVβ ‘off panel’: CD4+, light grey; CD8+, dark grey). (B) OCI-flow of CADM1+CD3+ cells versus OCI-flow of CADM1−CD3+ cells. Statistical analysis: Kruskal-Wallis test with Dunn post-test, 95% confidence interval (CI). * denotes p<0.05, *** denotes p<0.001. (C) Comparison of LM-PCR/HTS data (n = 28 ATL patients) and CADM1/TCRVβ flow cytometry. Statistical analysis: Spearman correlation.
Fig 2.
Expression of candidate ATL cell surface markers by the dominant TCRVβ-expressing population.
Staining was performed as described in Fig 1. (A) Representative flow cytometry plots of total live CD3+CD4+ cells from an ATL patient (LHN) and an AC (HHD). Plots display the most frequently expressed TCRVβ subunit in the respective donor. (B) Expanded clones are CCR4+CD7−CADM1+. Live CD3+CD4+ T cells from ATL patients (n = 21) with an OCI-flow (CADM1+CD3+) >0.7 were gated on the basis of expression of the dominant TCRVβ (designated TCRVβX+ or TCRVβX–). Total live CD4+ T cells from PBMC of n = 24 individuals without malignancy (patients with HAM or ACs) were included as controls. Whiskers represent maximum and minimum values. Statistical analysis: Kruskal-Wallis test with Dunn post-test, 95% confidence interval (CI). * denotes p<0.05, ** denotes p<0.01, *** denotes p<0.001.
Fig 3.
OCI-flow of CADM1+CD3+ cells is an excellent diagnostic test for monoclonal integration.
Receiver operator curves illustrating the specificity and sensitivity by which the OCI-flow of CADM1+CD3+ cells, the frequency of CD7−CD4+ or the frequency of CD25+CD4+ cells discriminate individuals with clinically evident ATL (n = 23) from individuals with non-malignant HTLV-1 infection (n = 24). Individuals previously diagnosed with ATL which were in clinical remission were excluded from this analysis.
Fig 4.
Expression HLA-ABC, CADM1 and Tax by CADM1+CD4+ T cells.
CD8+ cells were depleted from PBMCs of 15 ATL patients with a dominant ATL clone detectable by TCRVβ staining, and 10 ACs. Cells were cultured for 18h, after which they were surface stained with a viability stain followed by antibodies specific for the most frequently utilised TCRVβ (VβX), CD3, CD4, CD8, CADM1, PD-L1, FoxP3 and HLA-ABC (S2 table; panels 3, 4, 6 and 7). Cells were then permeabilised and stained with antibodies specific for Tax and FoxP3 and analysed by flow cytometry. Cells from ATL patients and ACs were gated on live CD3+CD4+CADM1+ cells which were positive or negative for the dominant TCRVβX as indicated. Intensity of expression of (A) MHC class 1 and (B) CADM1. (C) Frequency of Tax expression by ATL clones. Taxhigh ATL clones are plotted in red, and Taxlow ATL clones are plotted in blue. Statistical analysis: Kruskal-Wallis test with Dunn post-test, 95% CI. * denotes p<0.05, ** denotes p<0.01, *** denotes p<0.001.
Fig 5.
Experimental design of cell survival assay.
PBMCs from each of 9 ATL patients with a dominant ATL clone detectable by TCRVβ staining were depleted of CD8+ T cells. The CD8− PBMCs (CADM1+CD4+, purple; CADM1−CD4+, yellow) were cultured overnight either alone or in the presence of autologous CD8+ cells at a range of ratios, after which cells were stained with a viability stain and antibodies specific for CD3, CD4, CD8, CADM1 and the TCRVβ subunit which was most frequently used in that individual (‘TCRVβX’). Cells were then permeabilised, stained intracellularly with anti-Tax antibody, and analysed by flow cytometry. Absolute cell counts of CD3+, CD4+ and CD8+ cells were performed in parallel.
Fig 6.
Cultured CD8+ cells can kill autologous malignant cells in some donors.
CD8 depleted PBMCs from ATL patients (n = 9) with a known dominant TCRVβ were incubated in the presence of ex vivo (A) or cultured (B) autologous CD8+ cells at the indicated E:T ratios. After 18h, the absolute number of viable ATL cells (live CD3+CD4+CADM1+TCRVβ+) was quantified by flow cytometry and used to calculate the proportion of the ATL clone which had been specifically killed in the presence of CD8+ cells. The proportion of Tax+ and Tax−CADM1+ TCRVβ− cells which were killed was calculated in the same manner. (C) Ex vivo CD8+ cell killing of Tax+CD3+CD4+CADM1+ cells, Tax−CD3+CD4+CADM1+ cells and CD3+CD4+CADM1− cells in ACs (n = 10). Subsets of cells which expressed Tax (in both ATL and ACs) are plotted in red.
Fig 7.
Tax-expressing cells are preferentially killed by cultured autologous CD8+ cells.
(A) Selective loss of live Tax-expressing ATL cells after incubation with cultured CD8+ cells. Extended analysis of data from Fig 6. ATL clones from three Taxhigh ATL patients (LGZ, LGC and LGB) were gated on the basis of Tax expression as shown. For each donor, graphs show the percentage of Tax+ or Tax−cells which were killed in the presence of cultured autologous CD8+ cells. Flow plots show Tax and CADM1 expression by live CD3+CD4+CADM1+TCRVβX+ cells from each individual after culture alone or in the presence of CD8+ cells at the highest E:T ratio tested. (B) Comparison of the rate at which ex vivo and cultured CD8+ cells supress survival of the populations indicated in ATL patients (n = 9) and ACs (n = 10). Subsets of cells which expressed Tax (in both ATL and ACs) are plotted in red. Data from Fig 6 was analysed by nonlinear regression to estimate the % change observed in each population with each 1% increase in CD8+ cells present in the co-culture. A negative rate indicates that number of viable target cells recovered from the co-culture was greater in the presence of CD8+ cells versus in the absence of CD8+ cells. Statistical analysis (CADM1+TCRVβ− groups only): Wilcoxon matched pairs test, two tailed, 95% confidence interval.