Expansion of functional personalized cells with specific transgene combinations.

Fundamental research and drug development for personalized medicine necessitates cell cultures from defined genetic backgrounds. However, providing sufficient numbers of authentic cells from individuals poses a challenge. Here, we present a new strategy for rapid cell expansion that overcomes current limitations. Using a small gene library, we expanded primary cells from different tissues, donors, and species. Cell-type-specific regimens that allow the reproducible creation of cell lines were identified. In depth characterization of a series of endothelial and hepatocytic cell lines confirmed phenotypic stability and functionality. Applying this technology enables rapid, efficient, and reliable production of unlimited numbers of personalized cells. As such, these cell systems support mechanistic studies, epidemiological research, and tailored drug development.

Polymicrobial sepsis and non-specific immunization induce adaptive immunosuppression to a similar degree.

Sepsis is frequently complicated by a state of profound immunosuppression, in its extreme form known as immunoparalysis. We have studied the role of the adaptive immune system in the murine acute peritonitis model. To read out adaptive immunosuppression, we primed post-septic and control animals by immunization with the model antigen TNP-ovalbumin in alum, and measured the specific antibody-responses via ELISA and ELISpot assay as well as T-cell responses in a proliferation assay after restimulation. Specific antibody titers, antibody affinity and plasma cell counts in the bone marrow were reduced in post-septic animals. The antigen-induced splenic proliferation was also impaired. The adaptive immunosuppression was positively correlated with an overwhelming general antibody response to the septic insult. Remarkably, antigen "overload" by non-specific immunization induced a similar degree of adaptive immunosuppression in the absence of sepsis. In both settings, depletion of regulatory T cells before priming reversed some parameters of the immunosuppression. In conclusion, our data show that adaptive immunosuppression occurs independent of profound systemic inflammation and life-threatening illness.

Peripherally induced Treg: mode, stability, and role in specific tolerance.

Foxp3-expressing regulatory T cells (Treg) have an essential function of preventing autoimmune disease in man and mouse. Foxp3 binds to forkhead motifs of about 1,100 genes and the strength of binding increases upon phorbol 12-myristate 13-acetate/ionomycin stimulation. In Foxp3-expressing T cell hybridomas, Foxp3 promoter binding does not lead to activation or suppression of genes which becomes only visible after T cell activation. These findings are in line with observations by others that Foxp3 exerts important functions in collaboration with T cell receptor (TCR)-dependent transcription factors in a DNA-binding complex. Tregs can be generated when developing T cells encounter TCR agonist ligands in the thymus. This process apparently depends on costimulatory signals. In contrast, extrathymic conversion of naïve T cells into Tregs appears to depend on transforming growth factor (TGF)-beta and is inhibited by costimulation. In fact, dendritic cell-derived retinoic acid helps the conversion process by counteracting the negative impact of costimulation. Tregs induced by subimmunogenic antigen delivery in vivo are much more stable than Tregs induced by antigenic stimulation in the presence of TGF-beta in vitro which correlates with the extent of demethylation of the Foxp3 locus. Tregs can be induced by conversion of antigen-specific T cells that occur with a very low frequency in wt mice. Conversion of naïve cluster of differentiation (CD)4 T cells into Tregs by a single peptide of HY antigens results in complete antigen-specific tolerance to an entire set of HY epitopes recognized by CD4 as well as CD8 T cells when presented with male skin or hemopoietic grafts.

Regulation of CD4+CD25+ regulatory T cell activity: it takes (IL-)two to tango.

Although CD4(+)CD25(+) regulatory T cells (Treg) represent a well-characterized population of T cells with in vitro and in vivo suppressive capacity, the basic mechanisms of suppression are still not understood. The constitutive expression of the high-affinity receptor for IL-2 has raised the question about the role of IL-2 in Treg function. Here, we review recent data indicating that IL-2 is not only necessary for the homeostasis of Treg but is also critical for the activation of Treg function. Since Treg do not produce IL-2 by themselves, their capacity to utilize IL-2 secreted by other T cells appears to be an essential component of Treg biology. This indicates that Treg suppressive activity is controlled by interaction with activated target cells via the soluble mediator IL-2. In Treg, IL-2 has been identified as a potent inducer of the immunosuppressive cytokine IL-10, an important mediator of Treg suppression in vivo. The efficient capture of IL-2 by Treg may, under conditions of limited IL-2 supply, cause IL-2 deprivation of responder T cells. This competition can explain some of the currently discussed discrepancies between in vivo and in vitro activity of Treg.