Periodic Report Summary - EBV HORIZONS (Heterologous immunity to Epstein-Barr virus: dissecting the role of cross-reactive CD8 T cells in mediating disease outcome in children, young adults and the el
(i) the role of convergent recombination in shaping the naïve T cell pool; and
(ii) the relationship between the composition of the naïve pool and clonotype selection into the memory pool.
The first study was carried out in mice to enable the greatest possible sampling of the naïve CD8 T cell pool on a genetically homogeneous background and inform downstream human studies. The naïve CD8 T cell pools of three mice were examined using high-resolution experimental and analytical techniques specifically developed for the purpose of repertoire deconvolution. TCR# sequences with convergent features were present at higher copy numbers within individual mice and were also shared between mice. These data demonstrated definitively that the naïve CD8 T cell repertoire comprises a hierarchy of recurrence rates for individual clonotoypes; this, in turn, is determined by relative production frequencies.
With these data in hand, we proceeded to examine the role of convergent recombination in human naïve and memory CD8 T cell repertoires. Large volumes of blood were collected from four healthy adult donors with robust immune responses to EBV and CMV, as verified by peptide-MHC class I tetramer staining. Polychromatic flow cytometry was used to isolate ultra-pure naïve and memory CD8 T cells, defined stringently on the basis of multiple phenotypic markers. The isolated cell populations were then sequenced using a high throughput platform (454 pyrosequencing) to characterise corresponding portions of the respective TCR repertoires. Both the extent of inter-individual TCR sharing and the degree of overlap between the naïve and memory compartments within individuals were determined by TCR clonotype frequencies, with higher frequency clonotypes being more commonly shared between compartments and individuals; TCR clonotype frequencies, in turn, were predicted by relative production efficiencies. Thus, convergent recombination shapes the TCR repertoire of the naïve and memory CD8 T cell pools, as well as their inter-relationship within and between individuals.
To date, this work has provided new insights into the mechanisms that shape the peripheral T cell repertoire. Overall, the project made significant strides towards unravelling the complexities of CD8 T cell immunity against an important human pathogen, Epstein-Barr virus, and training Dr Quigley to develop an independent research programme dedicated to the study of medically important viral infections in humans.