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

Oligonucleotide sequences^a.

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

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

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

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

Immunohistochemical characterization of human vascularized BME-rich organoids.

<p>Immunohistochemical characterization of explanted human vascularized BME-rich organoids using anti-CD34 (species specific: human and mouse; [39]), anti-CD45, anti-F4/80, anti-neutrophils, anti-vimentin and anti-α-SMA antibodies (Table S3). Cell nuclei were counterstained with hematoxylin. 10x and 40x images are shown.</p

Immunohistochemical characterization of control BME-rich organoids.

<p>Immunohistochemical characterization of explanted control BME-rich organoids using anti-CD34 (species specific: human and mouse; [39]), anti-CD45, anti-neutrophils, anti-vimentin and anti-α-SMA antibodies (Table S3). Cell nuclei were counterstained with hematoxylin. 10x and 40x images are shown.</p

Generation of BME-rich organoids with functional human blood vessels in immunodeficient mice.

<p>(a) A mixture of HUVEC^FLuc (3 x 10⁵) and human MSC (7.5 x 10⁴) were embedded in supplemented BME (150 µl) and inoculated subcutaneously in the ventral area (right lower quadrant) of immunodeficient mice. Human vascularized BME-rich organoids were easily identified as small bumps under the skin at all times <i>in vivo</i>. (b) Ventral D-luciferin-based F^Luc-BLI images of a representative mouse 1, 2, 4, 7, 10 and 13 weeks after implantation of the human vascularized BME-rich organoid. (c) Low magnification (4x) micrograph displaying the entire organoid (Scale bar is 1 mm). Hematoxylin and eosin (H and E)-stained sections taken from different part of the organoids revealed the presence of numerous luminal structures containing erythrocytes (arrowheads), and some glomeruloid microvascular proliferations (arrows). 10x and 40x images are shown.</p