| Abstract |
The asymptomatic phase of HIV-1 infection is characterized by a progressive depletion of
uninfected peripheral effector/memory CD4+ T cells that subsequently leads to immune
dysfunction and AIDS symptoms. Nevertheless, the precise mechanism of by which the virus
depletes uninfected CD4+ T cells, the so called bystander effect, remains elusive. Previous
studies of our research group suggest that proteins of the viral envelope participate in an
“immune-viral” synapse between HIV-1 infected macrophages and CD4+ T cells during antigen
presentation. At the synapse, the third hypervariable (V3) domain of HIV-1 envelope
glycoprotein gp120 interacts with the chemokine receptor CCR5 inducing an accelerated and
enhanced type of apoptosis, called activation-induced cell death (AICD), of the responding CD4+
T cells. It was also demonstrated that the V3-CCR5 interaction is ionic and it is governed by
positively charged residues of the V3 peptide and negatively charged peptides on the amino
terminal domain of CCR5, the impetus of this interaction being dependent on the charge of V3.
It was also shown that synthetic V3 peptides can inhibit the AICD of CD4+ T cells, but also
prevent the infection of primary cells by ΗΙV-1.
The present study aimed at the unveiling of the initial signaling molecules triggered by the
interaction of V3 epitope with the CCR5 receptor which lead the responding CD4+ T cells in
immune dysfunction of and the elucidation of the role of V3 epitope in the phenomenon of
AICD of uninfected CD4+ T cells. In order to reproduce the biological phenomenon in vitro, we
exposed macrophages to linear synthetic lipopeptides from the crown of V3 presented on
liposomes and then we induced antigen presentation complex formation with CD4+ T cells via a
superantigen presentation system. The impact of the HIV-1/V3 epitope on the transcriptional
profile of a CD4+ enriched T cell population during antigen presentation was determined by
oligonucleotide microarrays. The microarrays analysis revealed that 440 genes were affected in
transcriptional level by the presence of V3 epitope; 378 were up-regulated, 18 of which for at
least 10-fold, and 62 were down-modulated. The most significantly up- and down- regulated
transcripts could almost entirely be categorized as related to cell cycle the transcriptional
regulation. Functional classification of significantly modulated genes and identification of
canonical pathways and functional gene networks analysis were performed by an Ingenuity
Pathways Analysis (IPA) platform and overrepresentation of functional ontologies by DAVID
Bioinformatics Resources. The statistically significant enriched categories of genes and
networks identified by IPA were related to cell cycle, gene expression, immune response,
infection’s mechanisms, cell growth, proliferation and the process of antigen presentation.
Interestingly, the CD28 signaling pathway was significantly subverted by the V3 epitope. Taken
together, these results provide evidence for a hyper-proliferative gene expression signature,
triggered by V3-CCR5 interaction in responding CD4+ T cells during antigen presentation that
could account for AICD of uninfected CD4+ T cells in HIV-1 infection.
As cell cycle was prominent among all classifications according to the bioinformatics analysis,
we further assessed the impact of V3 epitope on the progression of cell cycle. The cell cycle
state of a CD4+ enriched population treated with the V3 epitope was characterized by flow
cytometry and an increase in the percentage of cells in S and G2/M phases was observed. The
protein levels of MKi67, which plays a pivotal role in cell proliferation, were evaluated by
fluorescence microscopy and the observed increase of MKi67 positive T cells in response to V3
confirmed the results of microarrays concerning the transcriptional up-regulation of MKi67. The
mRNA expression of CCNB1 gene, which encodes for a regulatory protein expressed in G2/M
phase and involved in mitosis, was also elevated in response to V3 suggesting that CCNB1 may
play a crucial role in cell cycle deregulation. Of special interest was also the striking upregulation
of NOC2L, which is novel gene that affects cell cycle progression regulating
transcription of p21 and inhibits apoptosis mediated by p53 and p63. The kinetic analysis of
mRNA expression shows that the up-regulation of the transcriptional levels was followed by a
sharp down-regulation, that could account for the observed cell death of uninfected CD4+ T
cells.
Interestingly, it was shown that V3 epitope also subverts CD28 signaling pathway, which is a
major driver of positive immune response eliciting sustained expression of IL2. Our studies
were focused on the role of the transcriptional factor NFAT5 (nuclear factor of activated T-cells
5) which exhibited extensive transcriptional up-regulation in response to V3 epitope according
to microarray analysis. The kinetic analysis of the mRNA expression of NFAT5 by real-time PCR
confirmed the increase in the transcriptional levels of NFAT5, and the enhanced protein
expression was confirmed by western blotting and flow cytometry. Assessing the functional
involvement of NFAT5, it was shown that cyclosporine A, which inhibits the NFAT activationr,
interfered with the proliferative promotion of V3 to CD4+ T cells supporting the role of NFAT5
in the V3 phenomenon. The evaluation of the transcriptional expression of IL2, which is
characteristic of the activation of CD28 signaling pathway showed enhanced expression of IL2
mRNA confirming the alternation of CD28 signaling pathway by V3 at functional level. Taken
together, our results suggest that during antigen presentation the V3 epitope impairs cell cycle
progression of CD4+ T cells promoting cells in S and G2/M phase by altering the transcriptional
levels of NOC2L, CCNB1, and MKi67 and subverts the CD28 signaling pathway, increasing the
transcriptional levels of NFAT5 and of IL2.
Due to the clinical relevance of our studies, there was a prerequisite to extend our findings in
HIV-1 infected individuals. In experiments performed at Dr Kaufmann’s laboratory at Ragon
Institute of MGH, Harvard & MIT in Boston, we assessed the transcriptional levels of selected
genes that emerged from transcriptome analysis of our in vitro model in CD4+ T cells of HIV
infected individuals with different disease status. The evaluation of the selected genes (NOC2L,
MKI67, CCNB1, CCND2, PDPK1, NFAT5, PI3KR1, PAK1, IFI6) in HIV-infected subjects at different
state of disease could provide valuable information on the immune components involved in
depletion of uninfected CD4+ T cells during HIV infection and enable us to identify the
intracellular pathways that are involved in the V3-induced cell death of uninfected CD4+ T cells.
A statistically significant reduction in the transcriptional levels of NOC2L was observed in HIV-1
infected individuals in comparison with healthy donors and NOC2L mRNA expression was
adversely correlated to viral load. It was also demonstrated that HIV-1 subjects had
significantly elevated MKi67 mRNA levels in relationship with HIV-1 negative individuals and
there is a significant direct correlation to viral load. Of interest, elite controllers exhibited
increased transcriptional levels of MKi67 and CCNB1 genes, which both encode for key
molecules in cell cycle progression. Taken together, these data suggest that the cell cycle
progression is impaired in HIV-1 infected individuals determining the disease stage.
V3 epitope re-immerged as a promising target for neutralizing antibodies when the 3D
structure was recently revealed. However, its precise role in HIV infection remains elusive. The
delineation of the molecular mechanism by which V3 epitope condemns the uninfected CD4+ T
cells to AICD is of major interest as it may account for progressive CD4+ T cell decline during the
asymptomatic phase. The findings of the present study provide evidence for a hyperproliferative
gene expression signature, triggered by V3-CCR5 interaction in responding CD4+ T
cells during antigen presentation. The evaluation of transcriptional levels of genes implicated in
V3 signaling in CD4+ T cells in HIV-infected patients of different disease status shed light on
genes involved in regulation of cell cycle. Unveiling the key participating molecules in
intracellular signaling within the responding CD4+ T cells may provide the impetus to design
novel therapeutic strategies such as the salvaging of uninfected T cells from HIV-induced AICD.
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