ARTICLE
Auteur(s) : François Villinger, Aftab A Ansari
Department of Pathology and Laboratory Medicine, Emory
University School of Medicine and Yerkes National Primate
Research Center, Atlanta, GA, USA
accepté le 25 Juin 2010
IL-12, a potent immunomodulator
Interleukin 12, originally termed the natural killer cell
stimulating factor was identified in 1990 by G. Trinchieri
et al. [1], as a heterodimer comprised of a p40 and a p35
subunit, covalently-linked, for the biologically active p70
cytokine. Besides activating NK cells, IL-12 was rapidly identified
as a master switch for differentiating naïve CD4+ T cells towards
the Th1 pathway, a pathway that is antagonized in the presence of
IL-10 [2]. Although IL-12 was initially purified from the
supernatant of a lymphoblastoid cell line [1], several studies
rapidly highlighted the monocytoid lineage, macrophages and myeloid
dendritic cells being the primary, physiological sources of IL-12
in response to a large variety of infectious agents that express
pathogen-associated molecular patterns (PAMPs) [3]. The triggering
of toll-like receptors (TLRs) by these PAMPs, effectively shapes
the developing immune response. The role of IL-12 in promoting the
generation of potent effector responses was highlighted by several
studies in mice. These demonstrated this cytokine's ability to
resolve or markedly limit infections that would otherwise linger or
remain chronic, such as leishmania, toxoplasmosis, listeria and
murine AIDS [4-7]. Interest in the application of such a potent
immune modulator in the treatment of various tumors was also
rapidly explored, secondary to its unique, dual biological effect
of enhancing both the innate and adaptive cellular effector
mechanisms along with its anti-angiogenic properties [8]. However,
toxicities and death resulted from an ill-fated, phase II trial for
the treatment of advanced renal carcinoma [9], which markedly
slowed the clinical applications of this cytokine, even though the
mechanisms underlying such toxicities have since been ascribed to
an inappropriate dose and administration schedule. Another source
of clinical caution stems from the fact that IL-12 shares its p40
and p35 chains with other members of the IL-12 family, IL-23 and
IL-35, leading to erroneous conclusions regarding the role of IL-12
in select autoimmune disorders or their models (e.g. inflammatory
bowel disease, experimental autoimmune encephalitis and others).
Studies using IL-23 are of particular interest since this cytokine
represents another master switch for a CD4 T cell subset
synthesizing IL-17, and is thus termed Th17. Results of
studies using IL-12 p40 antagonists in a variety of autoimmune
disease models led to the conclusion that the perceived deleterious
effects of IL-12 need to be revised since the etiology of the
inflammatory response were primarily ascribed to IL-23 [10].
IL-12 and HIV
One of the hallmarks of HIV infection is an early immune
dysfunction characterized by the gradual erosion of cellular
effector responses, even prior to extensive CD4+ T cell loss.
Although the mechanisms leading to such immune dysfunction are
multi-factorial, many studies focused primarily on the function of
CD4+ T cells since the CD4+ T cells are the primary target of virus
infection. Results from several studies of antigen-specific immune
responses conducted on a variety of patient groups published in the
nineties, led to the conclusion that both CD4+ and CD8+ T cell
responses were markedly enhanced ex vivo by the addition of IL-12
[11-13], but that such a capacity to respond decreased in patients
with marked CD4 loss and disease progression [14]. The enhancement
of CD4+ T cell antigen-specific responses was boosted further in
HIV-infected patients by the demonstration that IL-12 inhibited
apoptosis in this cell lineage [15], suggesting a potential,
prolonged effector function in addition to enhanced magnitude of
response. The benefit of IL-12 therapy was tested and confirmed in
vivo using the non-human primate model of AIDS using the Indian
rhesus macaques infected with simian immunodeficiency virus (SIV)
[16, 17]. In both studies, viral loads did not appear affected by
the therapy, even though IL-12 was administered in the absence of
antiretroviral therapy, and a marked increase in the frequency of
circulating NK cells and partial restoration of NK lytic functions
were noted. Our study also highlighted partial restoration of
SIV-specific cytotoxic responses during the chronic SIV infection
period [17]. In contrast, IL-12 therapy administered at late stages
of SIV infection characterized by low CD4 T cell levels and
opportunistic infections showed that most of the cytokine responses
were lost during the late stage of the disease, confirming the data
obtained with samples from HIV patients analyzed ex vivo [14].
Analysis of the mechanisms regulating the IL-12 response
potential in PBMCs from HIV infected patients was also conducted,
with several reports highlighting a marked diminution in IL-12
production by monocytes, macrophages and dendritic cells from
HIV-infected patients upon stimulation with a variety of agents
[18-21]. Such findings account for the early dysfunction and loss
of cell-mediated responses to HIV and other pathogens long before
CD4 T cells numbers have decreased to levels associated with AIDS.
While such a reduction in IL-12 production may be secondary to
direct infection of monocytes/macrophages or dendritic cells, the
relatively low frequency of such infected cells strongly argues
against a direct effect of the virus. However, two independent
studies uncovered at least two distinct pathways elicited by HIV
proteins to inhibit IL-12 production by the monocytic lineages [22,
23]: activation of monocytes/macrophages with Staphylococcus aureus
in the presence of HIV gp120 resulted in a skew in the cytokine
profile produced: IL-12p40 mRNA was markedly diminished while IL-10
production was upregulated, effectively inhibiting IL-12 protein
production. The second mechanism is mediated by extracellular HIV
vpr, which acts as a glucocorticoid receptor co-activator and
represses the production of IL-12 p35 production and release of the
biologically active IL-12 heterodimer following activation of
monocytic lineages, in a dose-dependent manner [23]. Both
mechanisms markedly diminish IL-12 production in response to
infection, and likely limit the generation of new and recall type I
immune responses.
This latter finding is critical when considering the need to
immunize HIV-infected patients and generate effective immune
responses, both against HIV and other pathogens, which may cause
opportunistic infections. With regards to HIV infection, data from
our laboratory have clearly demonstrated the potential of IL-12 in
markedly altering the course of disease. Rhesus macaques
pre-conditioned with IL-12 were inoculated intravenously with the
highly pathogenic SIVmac251. While the magnitude of the acute viral
loads did not differ markedly between IL-12-treated and untreated
control monkeys, IL-12-treated monkeys established potent immune
responses during the chronic phase capable of maintaining viral
load set points substantially lower than control monkeys [24]. The
monkeys given IL-12 at the time of infection also maintained a
disease-free status for three to four years, while untreated
SIV-infected monkeys developed AIDS and had to be euthanized
between six to twelve months post-infection. These findings
underscore the potency of IL-12 in profoundly shaping immune
responses following the initial antigen encounter, markedly
altering the host pathogen interactions in vivo, an observation
seen in murine AIDS [4], simian AIDS [24] and simian malaria
[25].
Thus, the use of IL-12 as an adjuvant to vaccines is amply
justified, although its incorporation into larger vaccine trials
has lagged, in large part due to the early mishap in the renal
carcinoma phase II trial [9]. Several studies have used IL-12 as an
adjuvant for a variety of immunizations in mice including HIV,
showing excellent efficacy. Studies in non-human primates however,
have highlighted the value of IL-12 as an adjuvant to DNA
prime/virus-like particle protein boost vaccines, where IL-12
administered in the prime and/or in the boost did not prevent
infection, but resulted in statistically significant reductions in
acute and chronic SIV viral loads (> 2 log), using a
challenge virus recognized as difficult to control [26]. Other
studies from the Wyeth Lederle team used plasmid DNA-delivered
vaccine alone with plasmid-delivered IL-12 and IL-15 [27]. These
studies showed improved cellular and humoral responses in the
IL-12-adjuvanted groups, as well as improved control of SHIV89.6p,
while monkey groups adjuvanted with IL-15 alone, behaved like the
animals administered the non-adjuvanted vaccine. In fact, the need
for IL-12 in generating HIV env-specific cytotoxic responses was
demonstrated more recently in IL-12KO mice in which supplementation
with exogenous IL-12 restored such responses [28].
Future of IL-12 use in the context
of HIV
As outlined above, IL-12 therapy in the context of HIV infection
may need to be revisited. Its most relevant application would be in
the context of opportunistic infections that are best met with type
I effector immune responses. In fact, IL-12 therapy is currently
administered to patients with Kaposi's sarcoma, for which other
therapies have failed [29, 30]. It has to be borne in mind though
that most opportunistic infections occur at late stages of HIV
infection, following extensive erosion of immune competency of the
host, a stage during which primate studies have shown limited
clinical benefit of IL-12 therapy alone. It may be possible to
explore combination therapies, using additional cytokines such as
IL-7, 15 or 21 and or other immune modulatory approaches,
to attempt to revive T cell effector responses. The use of ART in
conjunction with IL-12 has also not been tested in this model.
Nevertheless, it is our belief that IL-12 may play a critical
role as an adjuvant to immunizations delivered to HIV-infected
patients. These patients suffer from suboptimal IL-12 production in
response to vaccine adjuvants and thus, exogenous, localized
delivery of cytokines would palliate the insufficient IL-12
production from dendritic cells and/or macrophages and ensure
appropriate T cell differentiation and recruitment. While the
investigation of IL-12 in the context of HIV was very active during
the nineties, the last decade has seen comparatively less activity.
The optimization of cytokine administration for select uses has
nevertheless made important progress, and it is likely that
targeted and localized administration of cytokines such as IL-12,
which have a relatively narrow therapeutic range, remains to be
fully explored. Thus, use of encapsulation in micro- or
nanoparticles, restricting the delivery to APCs and/or the
co-delivery of IL-12 with an antigen delivered via transducing
vectors, are likely to be more effective than free cytokines and
far better tolerated.
Thus, we believe that, primarily as an adjuvant to vaccines,
IL-12 remains one of the most promising candidates both for
HIV-infected patients and developing countries where endemic
helminth infections limit the production and release of endogenous
IL-12 by resident APCs, leading to suboptimal immune responses to
select pathogens.Disclosure. None of the authors has any
conflict of interest or financial support to disclose.
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