PhD SCIENCE REPORT Rebekka Geiger

Rebekka Geiger

I started my Ph.D in the lab of Federica Sallusto, Ph.D at the Institute for Research in Biomedicine, Bellinzona in November 2005. During the first part of my Ph.D I was mainly focused on the development of a novel assay to examine the repertoire of human naïve CD4+ and CD8+ T cells. The second part was then devoted to the validation and application of this method. After 3 ½ years of working on that project, I plan to defend my Ph.D next spring (April 2009).

During vaccinations or in the course of natural infections a small number of antigen-specific T cells expands and differentiates into effector cells. Of these cells only a small proportion will give rise to memory cells providing livelong protection. To better understand the outcome of antigen-specific responses we were interested to study more precisely the composition of the naïve pool. However, due to their low number and high activation threshold, a reliable technique to assess the specificity and frequency of human antigen-specific naive CD4+ T-cells is still missing. We established a method that is circumventing these limitations. The protocol is mainly based on a polyclonal amplification step. In particular, a large sample of naïve T cells is divided into several replicate cultures that are individually amplified with high efficiency and fidelity through polyclonal activation. Each culture, representing a set of expanded T cell clones, is then tested for the presence of antigen-specific cells in conventional proliferation assays using monocytes or MoDCs as APCs. The frequency of specific T cells in the naïve pool is estimated from the number of positive cultures. Specific T cell clones can then be isolated from positive cultures for further analysis. Using this method, we were able to assess the frequency of cells specific for KLH, PA, TT, DerpI, PPD and CMV. We found that frequencies of antigen-specific T cells in the naïve pool were very high, ranging from 1 to 100 per million naïve T cells, depending on the antigen and donor analyzed. The same experimental approach was adapted to the analysis of the memory T cell repertoire. Estimated frequencies in the memory pool were in general 5-100 fold higher and varied with the antigenic experience of the individual. Furthermore, T cells in the naïve pool showed a broad range of avidities compared to cells in the memory pool that were enriched in high avidity T cell clones. In conclusion, our results provide evidence that “affinity maturation” in T cells is achieved by selection of high avidity T cells in the memory repertoire. Thus, the method not only allows to determine the frequency, epitope specificity and identity (TCR sequence) of antigen-specific T cells in the human naïve T cell pool, but also provides the possibility to directly compare frequencies of naïve and memory T cell subsets. Experiments to examine the repertoire of CD8+ T cells were performed accordingly. High frequencies were detected for the model antigen MelanA/Mart-1, but only few positive cell lines could be identified using HIV and CMV peptides or peptide pools. We explained this observation with the fact that whole protein antigens were used for detection of CD4+ T cell responses, but only single peptides for CD8+ T cells, assuming that a higher number of epitopes is generated from a whole protein. Taken together this study provides a new method to monitor the human immune response to a large panel of antigens, especially vaccines as well as a way to exploit the large and pluripotent repertoire of naïve T cells to generate antigen specific T cell clones with high expansion potential, desired avidity and functional activity for cellular immunotherapy.

To continue in research, I would like to do a post-doctoral training abroad. During that time I would like to broaden my knowledge of techniques (confocal microscopy, immunohistochemistry etc.) and work on different cell types.

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