Fig. 1.

Bioengineering of D-CAR⁺ T cells. (A) Components of D-CAR. The extracellular (Ec) sugar-binding domain of human Dectin-1 was fused in frame to a modified human IgG₄ hinge and Fc region and the transmembrane (Tm) and cytoplasmic (Cyto) domain of human CD28, and Cyto CD3-ζ. HA₃ and FLAG₃ epitope tags were fused on the C and N termini, respectively. (B) Numeric expansion of T cells cocultured with aAPC clone #4 preloaded with Dectin-1–specific mAb in the presence of soluble IL-2 and IL-21. Data are plotted as mean ± SD from three different donors. Upward arrows indicate the addition of γ-irradiated aAPC. (C) Expression of D-CAR on CD4⁺ and CD8⁺ T cells after electroporation and propagation for 35 d. CD19-specific CAR⁺ T cells from the same donor were used as a gating control. (D) Comparative gene-expression profiles of the expanded D-CAR⁺ T cells and circulating T cells from healthy donors. Abundance of mRNA transcripts was measured by digital gene expression using the nCounter analysis system. (E) D-CAR⁺ T cells propagated for 35 d were costained with anti-CD3 and anti–Dectin-1 mAbs, in addition to staining with anti-CD45RA, CD45RO, CD28, CD62L, and CCR7, and were analyzed by flow cytometry. D-CAR⁺CD3⁺CD45RA⁻RO⁺ T cells were gated for expression of CD62L, CD28, and CCR7 to define central memory T cells.