Lymphocyte transformation test with CFSE
ROC curves were performed in CD3+cells for both C-LTT and dDC-LTT. The area under curve (AUC) was 0.6045 (p=0.1283) and 0.7826 (p<0.0005) and we selected a cut-off of 2.22 and 1.28 for C-LTT and dDC-LTT respectively, both with a specificity of 85% (Figure S2A).
The C-LTT with PBMCs showed a significant lower sensitivity (33.3%) compared with dDC-LTT (65.2%) (p=0.026). When we combined the results of C-LTT and dDC-LTT, no increase in the sensitivity was observed (65.2%) compared with dDC-LTT alone. Nevertheless, its specificity reduced to 82.6% (Figure 1A).
As dDC-LTT showed higher sensitivity than C-LTT, we focused on this test to evaluate the different cell subpopulations proliferation. Significant higher proliferations were obtained for all cell subpopulations from allergic patients comparing with healthy controls (Figure 1B). Moreover, in allergic patients, we observed significant higher proliferation in CD4+Th1 cells, compared with other cell subpopulations including CD4+Th2, CD8+ and NK cells.
When we analysed the results in terms of positivity, the sensitivity of dDC-LTT in CD3+cells increased from 65.2% to 73.91%, and to 82.6% when CD4+Th1 and NK cells were respectively included in the analysis and to 86.9% when the three cell subsets were analysed together (Figure 1C). Regarding specificity, it was similar for all cell subpopulations mentioned above (85%). Moreover, although the sensitivity increased to 91% with the inclusion of CD8+ cells, the specificity reduced to 80% (Figure 1C).
No correlation was observed between proliferation results and the time interval between drug administration and onset of symptoms or the time interval between the onset of symptoms and the performance of LTT (data not shown).