Morton, Laura T. (2011)
M.Res. thesis, University of Birmingham.
EBV-specific CD8+ T cells have previously shown to be present in high frequencies within the tonsil compared to the peripheral blood of healthy EBV carriers. The anatomical location of these EBV-specific CD8+ T cells in the tonsil is currently unknown and could be the means of identifying lytically infected cells. Class I tetramer staining can now be adapted for in-situ staining of EBV-specific CD8+ T cells.
We have utilized Quantum dot (Qdot) nanocrystal technology to form EBV-specific Qdot multimers. Each multimer has a potential to form strong MHC-TCR interactions and are extremely photo stable. Here, we have optimized and validated the use of EBV-specific Qdot multimers in-situ staining to visualize EBV-specific CD8+ T cells in human tonsil sections from healthy EBV carriers.
Saturation of streptavidin conjugated Qdot655 (StpQdot655) was achieved at a ratio of 3.9μg of MHC class I monomer to 1pmol of StpQdot655 forming EBV-specific Qdot multimers. In all experiments EBV-specific Qdot multimers were shown to be extremely stable and demonstrated specific binding in flow cytometry and microscopy analysis. Qdot multimers also showed comparable staining intensity to conventional tetramers in flow cytometry analysis of multiple T cell populations. Optimized confocal microscope settings, using Qdot655 and Alexa488 reagents, allowed for the visualization of EBV-specific CD8+ T cell clones using corresponding EBV-specific Qdot multimers and anti-CD8 Alexa 488 antibodies.
The use of EBV-specific Qdot multimers in an optimized staining protocol has now been validated for the visualization of EBV-specific CD8+ T cell via confocal microscopy. Once an EBV+ HLA matched tonsil specimen can be obtained the localization of EBV-specific CD8+ T cells in the tonsil can for the first time be determined.
CD4+ T cells play a central role in the immune response and their role in the control of EBV infection has become of increasing interest in recent years. Advances in class II tetramer technology has allowed the discovery of a unique phenotype of EBV-specific CD4+ T cells in IM donor blood and throughout IM resolution, cells begin to show an up regulation of lymphoid homing markers, CCR7 and CD62L. This suggests EBV-specific CD4+ T cells may be homing to the tonsil, the potential organ harboring EBV. For the first time, EBV-specific CD4+ T cell frequencies and their phenotype can be analyzed using an internally optimized class II tetramer staining protocol.
Here, we have shown an enrichment of EBV-specific CD4+ T cells in the tonsil of healthy carriers compared to the blood. These cells share the same phenotype as the total CD4+ T cell population in both blood and tonsil preparations. We have discovered a novel phenotype of EBV-specific CD4+ T cells in which the majority show CCR7+CD45RA+ expression, characteristic of a naïve population. These cells are clearly not naïve and the necessity to explore new memory markers to characterize EBV-specific CD4+ T cells is imminent.
This project shows exciting results indicating a clear importance of CD4+ T cells in the control of EBV infection and with the use of class II tetramers will eventually enable their exploitation as immunotherapeutic options for disease.
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