Single cell studies of human T cell function with applications for HIV-1

Abstract

T cells are one of the body’s main defenses against viruses and cancers and are therefore considered to play a major role in immunotherapies after stem cell transplantation and in HIV-1 vaccine and cure strategies. However, malignancies and chronic viral infections, such as HIV-1, eventually cause the T cells to become dysfunctional, resulting in a loss of control. As the T cell population is highly heterogeneous, studying the characteristics of the relatively few cells that are specifically recognizing infections and malignancies is of immense importance to fully understand what makes up an effective T cell response. Therefore, the aim of this thesis has been to evaluate the single-cell characteristics of T cells using microwell-chip based imaging and multi-color flow cytometry.

The cytokine IL-2 is widely used to expand T cells for immunotherapy, but it also leads to expansion of the regulatory T cells (Tregs) that dampen the desired T cell response. We evaluated the effects of addition of IL-7, which previously was shown to decrease Treg expansion, to the protocol by monitoring the actions of T cells with single-cell resolution in a microwell chip. Addition of IL-7 did not affect the migration properties or cell-cell interactions, however it increased T cell survival in the microwell chip. Overall, the microwell chip was proved to be suitable for T cell studies and addition of IL-7 was confirmed to be beneficial when preparing T cells for immunotherapy.

During HIV-1 infection, CD8+ T cells become exhausted, i.e. upregulate inhibitory receptors in a process linked to the loss of functional properties, due to the constant antigen burden. We found that the transcription factors T-bet and Eomes were inversely expressed on bulk and HIV-specific CD8+ T cells. Cells with a high expression of Eomes and a low expression of T- bet were linked to an increased expression of inhibitory receptors, a transitional memory phenotype and a loss of functional capacity. This transcriptional profile remained after more than 10 years of treatment, suggesting that this might contribute to an inability of HIV- specific CD8+ T cells to control the infection even during successful treatment.

We continued by investigating the role of a novel inhibitory receptor, T cell immunoglobulin and ITIM domain (TIGIT), on CD8+ T cell exhaustion during HIV-1 infection. TIGIT was upregulated on bulk and HIV-specific CD8+ T cells and was linked to an increased expression of markers of exhaustion and immune activation as well as a T-betdimEomeshi transcriptional phenotype. Furthermore, upregulation of TIGIT on HIV-specific CD8+ T cells was linked to the downregulation of its complementary co-stimulatory receptor CD226 and a diminished functional capacity. Finally, expression of the TIGIT/CD226 ligand PVR was upregulated of T follicular helper cells, representing a major source of latent and productive HIV-1 infection. The result suggests that PVR is upregulated on HIV-infected cells and provides another major obstacle for HIV cure strategies.