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
The main objective of this project was to characterise the role of cross-reactive CD8 T cells in the pathology of Epstein-Barr virus (EBV) infection in children, adults and elderly individuals. Work conducted earlier during this project characterised in detail the clonotypic repertoire of human CD8 T cells specific for Epstein-Barr virus (EBV) and cytomegalovirus (CMV) in healthy adults. This work also involved the successful establishment of a panel of EBV-specific CD8 T cell clones that were used for combinatorial library screening during the final year of the project to identify cross-recognised ligands without bias. These studies clearly showed that inter-individual sharing of T cell receptor (TCR) clonotypes occurs within antigen-specific CD8 T cell populations. A mechanism termed convergent recombination was proposed to explain this phenomenon, according to which TCR sharing between individuals is a function of production frequency during V(D)J recombination. On this background, we hypothesised that shared TCRs might be more cross-reactive and play an important role in the pathology of chronic viral infections. Accordingly, two major pieces of experimental work were conducted to address:
(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.
(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.