LTT in different clinical manifestations
ROC curves were performed for both C-LTT and dDC-LTT for the most frequent clinical entities, SJS-TEN, MPE, and AGEP to select the cut-off for positivity. In SJS-TEN patients, comparisons between both LTTs showed differences with an AUC of 0.78 (p=0.01) for C-LTT and of 0.96 (p<0.0001) for dDC-LTT (Figure S2B). Using a cut-off 2.2 for C-LTT and 1.28 for dDC-LTT, the sensitivity was 62.5% and 87.5% respectively, with 85% of specificity for both (Figure 2). In MPE patients, after ROC curve analysis, we stablished a cut-off of 1.71 (AUC=0.559, p=0-50) for C-LTT and 1.28 (AUC=0.603, p=0-28) for dDC-LTT, obtaining a specificity of 85% in both cases (Figure S2C). Results showed lower sensitivity in C-LTT, 14.3%, than in dDC-LTT, 41.7% (Figure 2). In patients with AGEP, ROC curves showed an AUC of 0.602 (p=0.45) and 0.854 (p=0.012) for C-LTT and dDC-LTT respectively. A cut-off of 2.28 for C-LTT and 1.28 for dDC-LTT was selected to a specificity of 85% in both LTT (Figure S2D). The sensitivity of C-LTT was only 33%, whereas in dDC-LTT it increased to 80% (Figure 2).
Taken into account the results obtained with dDC-LTT, we observed a significant higher proliferation in SJS-TEN patients compared with MPE patients (p=0.001) (Figure 2A). We also obtained a significant higher percentage of positive cases in SJS-TEN and AGEP patients, 87.5% and 80% respectively, compared with MPE patients (41.6%) (p<0.05 in both cases) (Figure 2B).
Afterwards, we analysed if the results in the proliferative response using dDC-LTT vary between different cell-subsets in these clinical entities. In SJS-TEN patients, the analysis of proliferation of different cell subpopulations showed significant higher levels of CD4+Th1 compared with CD4+Th2 cells, NK cells, mainly for the inflammatory subpopulation (p<0.0001, p<0.01, and p<0.01 respectively), but not compared with cytotoxic NK cells. Moreover, the proliferation of CD3+, CD4+, and CD8+ cells was also significantly higher than CD4+Th2 cells proliferation (p<0.01) (Figure 3A). Regarding MPE patients, the proliferation of CD4+Th1 cells was again significantly higher (P<0.001) than the rest of cell subpopulations analysed except for CD4+Th2 cells and NK with inflammatory pattern, CD3-CD56+IFNγ+(Figure 3B). Regarding AGEP patients, we observed a significant higher proliferation of CD4+Th1 cells compared with the general population of CD4+(P<0.01), CD4+Th2 (P<0.01) and cytotoxic NK cells (P<0.01) (Figure 3C).
According to the cut-off previously described for each clinical entity, we analysed the proliferative response in terms of positivity (Figure 3D-F). In SJS-TEN patients, despite the high sensitivity in CD3+cells (87.5%), when we included the results from other cell subpopulations, we were able to detect all patients (100%), concretely, when including CD4+Th1 and NK cells without reducing LTT specificity (Figure 3D). When we analysed the sensitivity of dDC-LTT in MPE patients combining CD3+ with the results of the most relevant cell subsets, there was an increase from 41.6% to 50% when including CD4+Th1 cells and to 58.3% with NK cells (Figure 3E). Moreover, when we combined the results of CD3+, CD4+Th1 and NK cells, the sensitivity increased to 66.7% without reducing the general specificity of 85%. In case of AGEP patients, the inclusion of CD4+Th1 in the analysis with CD3+ cells, did not improve the sensitivity (80%) (Figure 3F), but it increased to 100% after the inclusion of NK and CD8+ cells with a specificity of 85% and 80% respectively. The positivity based on the proliferation of CD4+Th1 cells is higher in the three clinical entities studied (Figure 4A). Nevertheless, the involvement of CD4+Th2 cells was higher in MPE patients (67%) compared with SJS-TEN (37.5%) and AGEP (50%). We also found differences in the percentage of positivity between inflammatory and cytotoxic NK cells, being higher for cytotoxic NK cells in SJS-TEN patients (87.5% versus 50%) (Figure 4B). On the contrary, in MPE patients the positivity of inflammatory NK cells was higher (71.4%) compared with the cytotoxic ones (28.6%). AGEP patients showed a more balanced proliferative response between inflammatory (80%) and cytotoxic (66%) NK cells.