| Abstract |
The human immunodeficiency virus (HIV) is responsible for the development of
the acquired immunodeficiency syndrome (AIDS). The hallmark of the HIV infection is
the depletion of memory/effector CD4/CD45RO T cells during the asymptomatic phase
that leads to the deregulation of the immune system. The exact mechanism of CD4 T
lymphocyte depletion is not well understood. Although several hypotheses have been
proposed to explain this aspect of HIV pathogenesis, the prevailing theory is that chronic
activation of the immune system by the HIV infection causes exhaustion of CD4 T cells
that die by activation induced cell death (AICD).
In order to investigate the mechanism by which HIV generates AICD to
memory/effector CD4/CD45RO T cells we developed an in vitro system using human
primary cells isolated from healthy donors. Since the major role of memory/effector
CD4/CD45RO T cells is to recognize antigens on antigen presenting cells (APC) and
regulate the appropriate immune response, we studied the pathogenic mechanism from an
immunological point of view. We investigated the effects of the third hypervariable
region (V3) of the envelope glycoprotein gp120 of HIV-1 during the antigen presentation
process. Using composite liposome particles with exposed V3 synthetic lipopeptides, we
demonstrated that during antigen presentation, when the V3 epitope was present on the
surface of APC, interacted at least with the chemokine receptor CCR5, causing enhanced
proliferation followed by sharp apoptosis of the responding T lymphocytes.
The main objective of the present study was to identify the intracellular signals
generated by the V3 phenomenon. The responding tetanus-specific memory
CD4/CD45RO T cells have an anticipated frequency in the order of 1 in 105-106 T cells.
This low frequency was considered unsuitable for the required intracellular signaling
studies and the V3 phenomenon had to be adapted using a more general antigen induced
activation system. For this reason the first part of this study dealt with the replacement of
the tetanus specific model by a superantigen (SEA) that activates up to 25% of CD4 T
cells to provide evidence that the V3 phenomenon occurred in the new system.
Proliferation and apoptosis studies indicated that in the presence of SEA, V3 generated
AICD. Phenotypic analysis of the cells confirmed that the main population affected was
the CD4/CD45RO T cells that express the chemokine receptor CCR5 and are reported to
be the target cell population of HIV during the asymptomatic phase. Removal of CD8
cells, as SEA activates both CD4 and CD8 cells, improved the observed V3 phenomenon
in terms of both clarity and repeatability indicating that this phenomenon is restricted to
CD4 cells. Addition of IL-2, but not IL-4, further improved the proliferation and the
apoptosis of the responding cells. PBMCs incubated with IL-2 in the absence of SEA
were also affected by the presence of V3 indicating a possible involvement of IL-2 in the
V3 phenomenon.
Further experiments were conducted in order to identify other cell surface
receptors for signal transduction. Our results showed an increase in the percentage of
cells expressing the IL-2 receptor, CD25, and the Fas receptor (CD95). Both receptors are
considered to be activation markers of antigen responding T cells and are involved in the
proliferation (CD25) and apoptosis (CD95) of T cells. In the same context, measurements
of intracellular calcium indicated that the presence of V3 increased the calcium levels at
time points where maximum apoptosis and Fas expression were observed. Proliferation
and apoptosis are dynamics states and although increase of intracellular calcium is
required for cell proliferation, sustained increase may also lead to AICD.
We finally investigated mRNA expression level changes in CD4 T cells after
exposing them to the above described SEA-V3 liposome system in relation to SEA-plain
liposome control. cDNA products were assessed by microarray analysis (Affymetrix
Human Genome U133 Plus 2.0 Array) with a panel of 47.500 transcripts of which 38.500
were genes of the human genome. Changes ≥2 (p≤0.05) were considered as significant.
Our results showed that 378 genes were up-regulated and 61 genes down-regulated. The
major clusters of these genes are involved in the activation of T cells and the increase in
intracellular calcium levels. Nuclear factor of activated T-cells 5 (NFAT5), which is a
transcription factor involved in the activation of T lymphocytes during antigen
presentation, showed an 18-fold increase in mRNA levels is considered to have a crucial
role in the activation of T cell induced by the V3 phenomenon. The microarray results
were further evaluated by real time PCR that verified the increase in the mRNA levels of
NFAT5. Up-regulation of CD38 gene, which is involved in increasing the intracellular
calcium levels, was verified by phenotypic analysis on CD4/CD45RO T cells that showed
increased levels of CD38 expression at time points where increased levels of intracellular
calcium and maximum apoptosis were observed.
The above results indicated a possible mechanism for the V3 phenomenon.
During antigen presentation, V3 peptides positioned on the surface of APC, interact with
at least the chemokine receptor CCR5 on the surface of CD4 T cells and generate AICD.
Because of the position of the V3 peptides on the surface of APC and the ionic
interaction between the V3 peptides and the CCR5 chemokine receptor, it is believed that
this interaction strengthens the contact between APC and T cells, and prolongs the
duration of antigen presentation. This generates enhanced activation signaling on T cells
with subsequent increase in intracellular calcium that leads to the activation of NFAT5
transcription factor. NFAT5 activates T cells leading to enhanced proliferation and
expression of activation markers such as CD25, CD38, CD95 and increased levels of
intracellular calcium. The abnormal enhanced activation produced by prolonged signaling
during the antigen presentation leads to the cell death of CD4 T cells by AICD.
Understanding the pathogenesis of HIV is crucial for the rational design of more
effective antiretroviral therapy. In this sense, the use of synthetic peptides with positively
charged amino acids that inhibited cell proliferation caused by the V3 phenomenon may
prove to be a useful tool for the battle against HIV. Additionally identification of the key
factors in the signal transduction system of the cells that are modulated by HIV may
assist in recognizing new therapeutic targets.
|
Search
