Exhaustion of T cells in HIV-1 and HIV-2 infection

HIV-1 and HIV-2 infection. The source virus for HIV-1 is SIVcp from
chimpanzees whereas HIV-2 originates from sooty mangabey (SIVsm). Pathogenicity
of HIV-2 is different from HIV-1, with the majority of HIV-2-infected patients
presenting with undetectable viral loads, no progression and mortality
comparable to uninfected individuals. This difference in pathogenesis can be
due to viral factors or anti-viral immunity. When HIV-1 and HIV-2 replication
and infectivity was compared, it was shown that there is no differences in
infectivity, however, HIV-2 replicates slower, therefore producing less virus
per infected cells. This reduced replication is independent of CTL (1).
Pro-viral DNA load in HIV-1 and HIV-2 infected individuals is also comparable
(2). Since HIV-1 and HIV-2 develop from different viral lineages, difference in
sensitivity to host restriction factors may play a role. Indeed, HIV-2 is more
susceptible to hTRIM5α-mediated restriction and this seems to be
capsid-mediated (3).
Exhausted HIV-specific CTL. Antigen-specific cytotoxic CD8+ T lymphocyte (CTL)
responses are considered to be major players in the protective immune response
against acute and chronic viral infections. In HIV-1 infection a key role for
CTL responses was indicated by several studies (4-9). CTL responses are also
mounted against HIV-2. That CTL may have a direct impact on HIV-2 replication
was indicated by the direct correlation between IFNγ+ HIV-2-specific CTL
responses and viral loads (10). However, several studies indicate a comparable
magnitude and breath of IFNγ-producing T cell response in HIV-1 and HIV-2
infection (11) indicating that it is not the numbers alone which explain
control of progression in HIV-2 compared to the loss of control in HIV-1.
Although the immune response in HIV-infected individuals can control HIV
replication for some time, ultimately, HIV-specific CTL in the majority of
HIV-1-infected patients and in a minority of HIV-2-infected patients fail to
clear or control HIV infection and AIDS develops. This failure of HIV-specific
CTL to eradicate or control long-term HIV raises the question whether
HIV-specific CTL become exhausted. Exhaustion of chronically antigen-stimulated
T cells was first described in a mouse model of chronic viral infection (12)
and is characterized by loss of proliferation, cytokine production and
cytotoxicity and the upregulation of inhibitory receptors (reviewed in (13)).
Similarly, CTL exhaustion has also been shown in HIV-1 infection (14-21).
Although most HIV-2 infected patients have undetectable viral loads, a recent
study indicated that even in aviremic HIV-2 infected patients viral replication
is ongoing (22). This would suggest that HIV-2-specific T cells are chronically
stimulated and this could lead to gradual exhaustion. Indeed, increased
expression of inhibitory receptors is found on HIV-2-specific CTL but it is not
clear whether they associate with viral loads (23, 24). The association of
inhibitory receptors with exhaustion in humans has largely been based on HIV-1
infection, therefore HIV-2 may provide a unique opportunity to investigate the
role of these receptors in a chronic infection that infrequently progresses. If
HIV-2-specific T cells show indeed an upregulation of inhibitory receptors but
are functionally not impaired, this would indicate that inhibitory receptors do
not necessarily reflect exhaustion but serve to dampen immune activation and
its detrimental effects. This also would suggest caution when equalizing immune
exhaustion, inhibitory receptor expression and immune dysfunction in HIV-1
infection. This study of T cell exhaustion in HIV-1 and HIV-2-infected patients
will allow to dissect the relation of inhibitory receptors and declining T cell
immunity in HIV infection.
Epigenetics and the regulation of exhaustion and T cell function.
Epigenetics is a means of DNA-sequence independent and reversible regulation of
gene transcription, leading to changes in chromatin structure through
methylation of DNA and modification of histones (25). Although T cell specific
transcription factors (e.g. Blimp-1, BATF, Eomes and T-bet) are implemented in
the regulation of T cell exhaustion and some factors like BATF may be directly
induced by inhibitory PD-1 receptor signaling (26), another mechanism involved
in exhaustion are epigenetic modifications. Several studies have demonstrated
that epigenetic modifications play an important role in T cell immunity (27,
28). However, epigenetic-mediated gene silencing was recently also linked to
CTL exhaustion by analyzing the epigenetic modifications of the inhibitory
receptor PD-1 (29). Thus epigenetic modifications can affect the normal
function of CTL but also potentially exhaustion. It is expected that different
classes of epigenetic modifiers would impact differently normal CTL function
and exhaustion. This is a question we will examine in this study.
HIV latency and epigenetics. Epigenetic modifications in the LTR region
of HIV also inhibit downstream HIV gene transcription, and control HIV
silencing in latency (30, 31). Since epigenetic modifications are reversible, a
number of epigenetic modifications or proteins that interact with these are
being targeted as therapeutic approaches to reactivate the latent HIV
reservoir. According to the *shock and kill* concept proposed in the last
years, reactivation of HIV provirus transcription by drugs would render the
latent reservoir visible and susceptible to the immune system. Combined with
cART this lead to the eradication of infection. Clinical trials are already
testing the effect of inhibitors of epigenetic modifiers on the latent HIV
reservoir. However, the effect epigenetic modifications have on the immune
system and specifically anti-HIV immunity is not well understood. Since
epigenetic modifications are genome-wide, their inhibition to reactivate the
HIV latent reservoir will also affect many cell types and genes. Few clinical
trials of epigenetic modifiers in HIV-infected patients analyzed the effect
these drugs have on HIV-specific immune responses, and these findings are
controversial (32). Our study will evaluate some of the most common inhibitors
to determine whether some of these have fewer deleterious effects on
HIV-specific CTL immunity or may even restore exhaustion.

1. Determine whether different classes of epigenetic modifiers affect
HIV-1-specific T cell immunity.
a. Investigate whether HIV-specific T cell exhaustion can be reversed by
inhibitors of different classes of epigenetic modifiers.
b. Examine whether inhibition of epigenetic modifiers affects the function of
HIV-specific T cells and other immune cells.
2. Determine whether HIV-specific T cell responses in HIV-2 infection are
exhausted.
We will examine the association between T cell exhaustion phenotype, function
and immune activation and test epigenetic inhibitors from objective 1 in HIV-2
infection.

This is an in vitro research study that uses peripheral blood received through
venipuncture from HIV-infected individuals. In vitro experiments are carried
out on isolated lymphocytes. Chronically HIV-1 or HIV-2-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 immediately or
cultured in vitro. For the epigenetic inhibitor studies PBMC will be treated
with different epigenetic inhibitors and subsequently analyzed. In both ex vivo
and in vitro studies, the phenotype of HIV-specific T cells (differentiation,
activation, inhibitory receptor expression and transcription factors), effector
function (cytotoxicity and cytokine production), proliferative capacity and
survival will be determined. For the epigenetic inhibitor studies the phenotype
and function of additional immune cell subsets (i.e. CD4+ and CD8+ T cells, B
cells, NK cells, macrophages and DC) will be measured.

Participation involves at least one blood donation of max 50 ml blood (5
tubes). The only risk for the volunteers regarding this study is the risk
connected to giving blood by using standard venipuncture. There is a rare, but
possible risk of infection, pain or bruising at the site of the needle stick.
Serious adverse events are extremely rare. Since this is an in vitro research
study, no benefits will arise for blood donors from taking part in this study.