Fig. 6: DN4.99 TCR-T cells do not recognize normal CD1c⁺ blood cells.

Primary DN4.99 TCR-transduced T (DN4.99 TCR-T) cells were assessed for killing in vitro normal circulating CD1c⁺ cells. a–c CD19⁺ B cells (a), CD14⁺ monocytes (b), and CD11c⁺ circulating dendritic cells (cDCs, c) were sequentially purified from the peripheral blood of healthy donors. Contour plots and histograms show the enrichment of each cell type and their CD1c expression (black histograms), determined ex vivo by flow cytometry labeling with the indicated monoclonal antibody (mAb). White histograms represent labeling with isotype-matched control mAb. Relative Fluorescence Intensity (RFI) was calculated as the ratio between the intensity of labeling of sample and control. d There was not any killing of primary B cells, monocytes and cDCs by DN4.99 TCR-T cells. T cells were co-cultured for 72 h with B cells, monocytes, or cDCs at a 1:1 Effector:Target (E:T) ratio, with either 20 μg/ml of blocking anti-CD1c mAb (gray bars), isotype control (black bars), or synthetic methyl-lysophosphatidic acid (mLPA; red bars), used as positive control. Target killing was determined by flow cytometry labeling and expressed as Elimination Index. e There was not any killing of primary B cells, monocytes and cDCs by DN4.99 TCR CD8⁺/CD4⁺ T cells. T cell co-cultures with B cells, monocytes, or cDCs and target killing assays were performed as described for primary leukemia blasts. Gray bars represent co-cultures with anti-CD1c mAb, and black bars with the isotype control. Note that DN4.99 TCR-T cells can only kill primary monocytes and cDCs upon supplementation with the synthetic mLPA-specific antigen. Data are represented as mean ± SD. **P = 0.0071; *** = 0.0001; not significant (ns) were determined by Ordinary one-way ANOVA followed by Tukey’s multiple comparison test. Shown are results obtained in independent experiments with n = 3 replicates/each displayed as mean; (d) n = 4 experiments for cDCs + mLPA; 5 for B cells + mLPA, monocytes + mLPA, cDCs + Isotype control and cDCs + anti-CD1c mAb; and n = 6 for all the other conditions. e n = 2 for cDCs and n = 3 for B cells and monocytes. Normal circulating CD1c⁺ cells were purified from different healthy donors in each experiment.