T cell tolerance

Thymocytes undergo a number of defined steps during their development towards mature T cells. Because of the random nature of the TCR rearrangement the positive selection checkpoint is necessary to ensure that the double positive (DP) cells can interact with the MHC molecule. Negative selection, which is important to eliminate self reactive thymocytes, is controlled by the avidity of the interaction between the TCR and the self antigen. This means that cells which interact with a low avidity to self antigen can survive and those with a high avidity die by apoptosis (negative selection). However, the exact molecular pathway of negative selection is not yet known. The basic idea of our approach is that we think that a transcription factor which is important for negative selection should be higher expressed in the DP state of the T cell development compared to the double negative and the single positive state. Preliminary microarray analyses identified, besides others, one gene as potential candidates of our approach. Because, there is not much known about it we wanted to investigate the importance in T cell development and followed two approaches: First, we placed its ORF under control of the CD4 promoter and generated a mouse line which overexpresses this particular gene in T cells specifically. Second, using the Cas9/CRISPR system we produced a knockout strain. As soon as we have finished analysing the founders both strategies are followed by deep analysis of T cell development which includes FACS analysis of different developmental stages as well as apoptosis and proliferation assays. The outcome which we would expect is massive apoptosis of peripheral T cells within the overexpression model and less negative selection of thymocytes in the knock out mouse model. Although, this is only a prediction, by following both approaches we should gain additional knowledge of the importance of our gene of interest during T cell development.

In addition, we investigated whether additional rounds of recombination occur during T cell development upon a negatively selecting signal. To test this hypothesis, we generated by multiple gene targeting a model in which a rearranged TCRα chain is expressed from within the TCRα locus similar to the product of a physiological recombination. In this model we showed that secondary recombination (editing) events cannot rescue developing T cells from negative selection (Buch et al., 2002). Enforcing expression initiation at the CD4+CD8+ stage the hypothesis of editing in T cell development was readdressed this year in a modification of the model, yielding the same negative result (Kreslavsky et al. 2013). In further experiments we used this insertion transgene to investigate another specific selection checkpoint (β- selection) during earlier thymic development (Croxford et al. 2008). Currently, we address in this model the reasons for premature expression of TCR transgenes. To investigate thymic development further, we generated another mouse strain, in which α-chain expression of the HY-TCR is made inducible. This mouse strain is currently a major research focus in the lab.

TGF-β1 is a pleiotropically active cytokine and deregulation of TGF-β signaling in T cells results in a deadly systemic autoimmune syndrome. In order to delete TGF-β receptor specifically in mature T lymphocytes we generated by knock-in a CD4-expressing-cell-specific, tamoxifen-inducible Cre-strain (CD4-CreERt2) and crossed it to a mouse strain carrying a loxP- flanked TGFβ receptor gene In the resulting offspring we failed, surprisingly, to observed a decrease in the numbers of regulatory T cells as previously described when TGF-β receptor was ablated by constitutive Cre expression. Also, in contrast to current knowledge, we did not detect a generalized autoimmune syndrome in these mice, thus overturning current thinking about the role of TGF-β in the immune system (Sledzinska et al. 2013). Currently we are working on describing the effects of TGF-β ablation on terminal T cell differentiation.