In Vitro Characterization and Enhancement of HIV-Specific CD8+ T Cells

Antigen-specific cytotoxic CD8+ T cell responses are considered to be major
players in the protective immune response against acute and chronic viral
infections. In infections with the human immunodeficiency virus (HIV) the key
role of CD8+ T cells was indicated by several studies. Although the immune
response in HIV infected individuals can control HIV replication for several
years, ultimately, HIV-specific CD8+ T cells fail to clear or control HIV
infection and AIDS develops. This failure of HIV-specific CD8+ T cells to
eradicate or control long-term HIV raises the question whether HIV-specific
CD8+ T cells become exhausted, leading to the functional loss of virus-specific
CD8+ T cells in chronic viral infections. Mechanisms regulating gene activity
include epigenetic modification and post-transcriptional regulations.
Epigenetic modification leads to chances in chromatin structure through
methylation of DNA and modification of histones. Epigenetic-mediated gene
silencing was recently also linked to CD8+ T cell exhaustion. Since epigenetic
modulations are reversible, inhibiting enzymes involved in epigenetic
modifications may represent a new approach to modulate the function of T cells
in viral infections. Post-transcriptional regulation is another mechanism
controlling the activity of genes. So far, microRNA, long noncoding RNA and
RNA-binding proteins have been indicated as altering gene expression in a
network by mechanism like silencing translation, destabilizing mRNA, preventing
degradation and modulating localization. Very little is known about the role
these post-transcriptional regulators play in immune responses and specifically
cytotoxic CD8+ T cells although dysregulated post-transcriptional regulators
may contribute to the exhausted phenotype of HIV-specific CD8+ T cells.
In this study, we will analyze the effect epigenetic inhibitor have on the
function of HIV- and CMV-specific CD8+ T cells. We will also characterize the
expression profile of several posttranscriptional regulators in HIV- and
CMV-specific CD8+ T cells from HIV-infected controls. We will take advantage of
having the ability to analyze two different virus-specific CD8+ T cells in the
same HIV-infected individual: HIV-specific CD8+ T cells are exhausted
virus-specific CD8+ T cells which fail to control chronic viral infection
whereas CMV-specific CD8+ T cells successfully control chronic CMV infection.
This will allow us to directly compare the effect of epigenetic inhibitors and
the expression of post-transcriptional regulators in these two CD8+ T cell
population and determine which will restore the function and behavior of the
failed HIV-specific CD8+ T cells to the level of the successful CMV-specific
CD8+ T cells.

1. To examine whether epigenetic inhibitors can reverse exhaustion of
HIV-specific CD8+ T cells.
2. To determine whether HIV-specific CD8+ T cells have altered expression of
post-transcriptional regulators.

This is a research study that uses peripheral blood received through
venipuncture from HIV-infected individuals. In vitro experiments are performed
on isolated lymphocytes. Chronically HIV-infected volunteers will be asked to
donate blood (maximum 50 ml, 5 tubes) either during their scheduled physician
visit or during a scheduled visit for the blood donation. PBMC will be isolated
from peripheral blood and either analyzed or cultured in vitro. For the
epigenetic inhibitor studies we will treat PBMC in culture with different
epigenetic inhibitors and then the phenotype and function of HIV-specific and
CMV-specific CD8+ T cells will be analyzed. The phenotype of HIV-specific CD8+
T cells will be determined by flow cytometry (memory differentiation, cytokine
production and inhibitory receptor expression) immediately after isolation or
after peptide stimulation. In some instances HIV-specific CD8+ T cells will be
peptide-stimulated and proliferation will be measured. In some experiments we
will examine the rate of cell death of HIV-specific CD8+ T cells. For the
post-transcriptional regulator analysis, we will sort HIV- and CMV-specific
CD8+ T cells from PBMC and analyze the expression of select
post-transcriptional regulators (miRNA, lncRNA and RBP) by RT-PCR and RNAseq.
For some of these post-transcriptional regulators, we will measure their
intracellular expression by flow cytometry when the assay is available.

There are no benefits for participants in this in vitro research study. The
only potential risk of participation in this study is the minor risk connected
to blood donation through venipuncture.