Oligonucleotide sequences^a.

a<p>Sequences of the various oligonucleotides applied for the construction of the vectors, and subsequent verification of vector sequences.</p

Effect of competitor populations on CIR-mediated T cell activation.

<p>CIR-positive Jurkatα^CEA-CIR-EGFP effector (E) cells were stimulated with CEA-positive target cells for 16 hours in the presence of increasing amounts of CIR-negative Jurkat competitor (C) cells. The expression of CD69 on E∶C ratios ranging from 100∶1 to 1∶100 was measured by FACS on pre-activation (left panels) and post-activation (right panels) mixtures.</p

CIR-mediated activation of human T cells.

<p>(A) Cell-surface expression of CEACAM5 (CEA) and NIP-modified molecules on HeLa, HeLa^CEA and HeLa cells labeled with 2.5 µg/mL of the hapten (HeLa^NIP). (B) FACS analysis of CD69 expression by Jurkat^EGFP, Jurkatα^CEA-CIR-EGFP and Jurkatα^NIP-CIR stimulated either with immobilized anti-CD3 mAb or target cells (E∶T = 1∶1; HeLa, HeLa^CEA or HeLa^NIP) for 16 hours.</p

CIR-mediated activation of human T cells.

<p>(A) Cell-surface expression of CEACAM5 (CEA) on HeLa, HT1080, MDA-MB-231 and MKN45 cells. (B) FACS analysis of CD69 expression by Jurkat, Jurkatα^CEA-CIR-EGFP and Jurkatα^NIP-CIR stimulated either with immobilized anti-CD3 mAb or target cells (E∶T = 1∶1; HeLa, HT1080, MDA-MB-231 or MKN45) for 16 hours.</p

Phenotypic and functional characterization of selected Jurkatα^CEA-CIR-EGFP cells.

<p>(A) Comparative analysis of EGFP and cell surface CIR expression of the initial (Jurkatα^CEA-CIR-EGFP) and the selected (Jurkatα^CEA-CIR-EGFP/2S) cell population after two rounds of activation/selection on CEA-positive target cells. (B) Comparative analysis of CD69 expression by Jurkatα^CEA-CIR-EGFP and Jurkatα^CEA-CIR-EGFP/2S cell populations after stimulation either with immobilized anti-CD3 mAb or target cells (E∶T = 1∶1; HeLa or HeLa^CEA).</p

Selection of CIR-activated T cells.

<p>Jurkatα^CEA-CIR-EGFP effector (E) cells and CIR-positive Jurkatα^NIP-CIR competitor (C) cells at a E∶C mixing ratio 1∶1000 were stimulated with CEA-positive target cells for 16 hours and further sorted on the basis of EGFP and CD69 expression. After a period of cell expansion the activation/selection cycle was repeated.</p

IL-2 production by Jurkat^EGFP and Jurkatα^CEA-CIR-EGFP cells stimulated either with plastic immobilized anti-CD3 mAb or target cells (E∶T = 1∶1; HeLa or HeLa^CEA) for 48 hours.

<p>IL-2 production by Jurkat^EGFP and Jurkatα^CEA-CIR-EGFP cells stimulated either with plastic immobilized anti-CD3 mAb or target cells (E∶T = 1∶1; HeLa or HeLa^CEA) for 48 hours.</p