Additive manufacturing enables the generation of 3D structures with predefined shapes from a wide range of printable materials. However, most of the materials employed so far are static and do not provide any intrinsic programmability or pattern‐forming capability. Here, a low‐cost 3D bioprinting approach is developed, which is based on a commercially available extrusion printer that utilizes a DNA‐functionalized bioink, which allows to combine concepts developed in dynamic DNA nanotechnology with additive patterning techniques. Hybridization between diffusing DNA signal strands and immobilized anchor strands can be used to tune diffusion properties of the signals, or to localize DNA strands within the gel in a sequence‐programmable manner. Furthermore, strand displacement mechanisms can be used to direct simple pattern formation processes and to control the availability of DNA sequences at specific locations. To support printing of DNA‐functionalized gel voxels at arbitrary positions, an open source python script that generates machine‐readable code (GCODE) from simple vector graphics input is developed.

The cancer-testis antigen NY-ESO-1 has been targeted as a tumor-associated antigen by immunotherapeutical strategies, such as cancer vaccines. The prerequisite for a T-cell-based therapy is the induction of T cells capable of recognizing the NY-ESO-1-expressing tumor cells. In this study, we generated human T lymphocytes directed against the immunodominant NY-ESO-1 157-165 epitope known to be naturally presented with HLA-A*0201. We succeeded to isolate autorestricted and allorestricted T lymphocytes with low, intermediate or high avidity TCRs against the NY-ESO-1 peptide. The avidity of the established CTL populations correlated with their capacity of lysing HLA-A2-positive, NY-ESO-1-expressing tumor cell lines derived from different origins, e.g. melanoma and myeloma. The allorestricted NY-ESO-1-specific T lymphocytes displayed TCRs with the highest avidity and best anti-tumor recognition activity. TCRs derived from allorestricted, NY-ESO-1-specific T cells may be useful reagents for redirecting primary T cells by TCR gene transfer and, therefore, may facilitate the development of adoptive transfer regimens based on TCR-transduced T cells for the treatment of NY-ESO-1-expressing hematological malignancies and solid tumors. Endogenously processed peptide epitopes recognized by autologous CD8 1 cytotoxic T cells and CD4 1 T helper cells have been identified for several class I and II molecules, respectively, pointing to the naturally occurring T-cell repertoire. 4,5 NY-ESO-1 expression in malignant tumors correlates with poor prognosis of the patients. 6,7 This might implicate that NY-ESO-1-expressing tumors do not easily escape from an NY-ESO-1-targeted therapy through the selection of Ag-loss variants as frequently observed after immunotherapies targeting the melanoma-associated differentiation Ags. 8,9 Following NY-ESO-1-directed immunizations specific T-cell responses can be elicited in the majority of patients with NY-ESO-1-expressing cancer. 2,3,10 However, there is no strong relationship between the amount of NY-ESO-1-specific T cells induced by the vaccines and the clinically observed tumor regressions. Several mechanisms may be responsible for this phenomenon: first, the levels of antigen-specific CTLs also capable of recognizing tumor cells may still be insufficient; second, the potentially tumor-reactive T cells may be silenced by tumor-mediated tolerance; third, the TCR avidity of NY-ESO-1-specific CTLs may be low because NY-ESO-1 is ectopically expressed in the thymus 11 and, therefore, high-avidity T cells are partly deleted.These problems may be solved with the adoptive transfer of NY-ESO-1-specific T cells, which display the combination of high TCR avidity, potent tumor recognition efficiency, and excellent proliferation capacity. The widespread application of autologous, antigen-specific T lymphocytes as a treatment method is limited due to the laborious procedure of T-cell isolation and characterization if tailored for every single patient. [12][13][14][15] This hurdle can be overcome if primary...

Bioprinting of engineered bacteria is of great interest for applications of synthetic biology in the context of living biomaterials, but so far, only a few viable approaches are available for the printing of gels hosting live Escherichia coli bacteria. Here, we develop a gentle extrusionbased bioprinting method based on an inexpensive alginate/agarose ink mixture that enables printing of E. coli into three-dimensional hydrogel structures up to 10 mm in height. We first characterize the rheological properties of the gel ink and then study the growth of the bacteria inside printed structures. We show that the maturation of fluorescent proteins deep within the printed structures can be facilitated by the addition of a calcium peroxide-based oxygen generation system. We then utilize the bioprinter to control different types of interactions between bacteria that depend on their spatial position. We next show quorum-sensing-based chemical communication between the engineered sender and receiver bacteria placed at different positions inside the bioprinted structure and finally demonstrate the fabrication of barrier structures defined by nonmotile bacteria that can guide the movement of chemotactic bacteria inside a gel. We anticipate that a combination of 3D bioprinting and synthetic biological approaches will lead to the development of living biomaterials containing engineered bacteria as dynamic functional units.

Bioprinting of engineered bacteria is of great interest for applications of synthetic biology in the context of living biomaterials, but so far only few viable approaches are available for the printing of gels hosting live Escherichia coli bacteria. Here we develop a gentle bioprinting method based on an alginate/agarose bioink that enables precise printing of E.coli into three-dimensional hydrogel structures up to 10 mm in height. Addition of a calcium peroxide-based oxygen generation system enables maturation of fluorescent proteins deep within the printed structures. We utilize spatial patterning with the bioprinter to control different types of chemical interaction between bacteria. We first show quorum sensing-based chemical communication between engineered sender and receiver bacteria placed at different positions inside the bioprint, and then demonstrate the fabrication of barrier structures defined by non-motile bacteria that can guide the movement of chemotactic bacteria inside a gel.