MHC multimer-binding CD8 T cells: Optimized detection, automated analysis and identification of antigen specific CD8 T cells in Narcolepsy type 1

Abstract

Antigen specific CD8 T cells are crucial mediators of specific immunity against intra¬cellular infections and cancer and when dysregulated can be involved in autoimmune diseases. Instrumental to the function of these cells, is the recognition of antigen presented in the context of MHC molecules on the surface of target cells, by the T cell receptor on the CD8 T cells. Disease relevant interactions between TCRs and peptide-MHC are complex and often of low affinity and frequency, complicating the detection of antigen specific CD8 T cells. In this thesis three different aspects of describing antigen specific CD8 T cells are touched upon: optimized detection using MHC multimers, automated analysis of MHC multimer-binding T cells and identifi¬cation of CD8 T cells with relevance to the autoimmune disease narcolepsy type 1, using DNA barcode labelled MHC multimers. In manuscript I, the impact of using different fluorochromes to detect virus specific CD8 T cells of both low and high frequency in MHC multimer assays, was investigated. We found that even though there was no effect on the ability to detect the T cell responses in question, the separation of MHC multimer-binding T cells from background events was affected by fluorochrome choice. This may be important for successful detection of low-affinity CD8 T cell populations such as those reactive towards self. Furthermore, we propose a bead-based strategy for optimization of fluorescence detection that is useful not only for MHC multimer assays, but for flow cytometry experiments in general. In paper II, the feasibility of using automated gating tools for analysis of MHC multimer binding T cells is shown. Increasing complexity of flow cytometry data has now surpassed our ability to analyze these data with standard manual gating strategies and has thus prompted the development of automated gating tools. The implementation of these tools in the broad scientific community has, however, been slow, mainly due to a gap in understanding of these tools. Therefore we tested the ability of three different algorithms, FLOCK, SWIFT and ReFlow to identify virus specific CD8 T cells of varying frequency. All tools performed well when detect¬ing MHC multimer binding T cell populations with frequencies above 0.1 % whereas SWIFT was the only tool to reliably detect populations below 0.02%. Furthermore, major challenges and obstacles for the integration of automated gating tools into the broad scientific community were identified and discussed. In paper III, the newly developed state of the art technology of DNA barcode¬labelled MHC multimers was used to detect neuron specific, self-reactive T cells with relevance to narcolepsy type 1 (NTl). NTl is a chronic, debilitating neurological sleep disorder. It is caused by the loss of neurons in the brain that produce the neuropeptide hypocretin, which is involved in regulation of wakefulness. The loss of hypocretin neurons is thought to be the result of an autoimmune attack, although this has never been indisputably proven. Rather, the autoimmune hypothesis is based on circumstantial evidence such as the very strong correlation between NTl and the HLA class II allele DQBl *06:02 and the increased NTl incidence that was observed after HlNl influenza vaccination in several countries. In our study, we investigate the ability of CD8 T cells from NTl patients and healthy controls to recognize a library of 1183 peptides from 7 proteins expressed by hypocretin neurons that are restricted to 8 different HLA types. What we find, is a broad presence of neuron-specific CD8 T cells in blood samples from NTl patients but also from healthy controls. We observed a difference in the number and frequency of these neuron-specific CD8 T cells between NTl patients and healthy controls positive for HLA-DQBl *06:02, indicating that the combination of expressing the risk HLA allele and also harboring a certain level of auto-reactive CD8 T cells might be important for disease development. Even though these findings do not provide conclusive evidence for the autoimmune hypothesis of NTl, they do for the first time show the existence of hypocretin neuron-specific CD8 T cells and are one step on the way to full elucidation of the pathogenesis of narcolepsy type 1.