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in Response to p24 Antigen in HIV-Infected Individuals: Potential Contribution of Anergy to HIV-Specific Unresponsiveness
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Laboratory of Immunoregulation and
Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, and
Warren Magneson Clinical Research Center, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in most,
but not all, individuals. These data suggest the possible contribution
of HIV-specific unresponsiveness to the lack of CD4+ T cell
proliferation to p24 Ag in HIV-infected individuals and that clonal
deletion alone does not explain this phenomenon. They also indicate the
potential for CD40LT and IL-12 as immune-based therapies for HIV
infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been suggested that the lack of enhancement of HIV-specific immune responses following initiation of effective antiretroviral therapy could be a result of clonal deletion, or severe depletion, of HIV-specific CD4+ T cells during the earliest stages of infection (21, 22). It is also possible that HIV-specific cells are present, but they are unresponsive. HIV-specific cells could be unresponsive due to specific tolerance to HIV Ags or to a global unresponsiveness as a consequence of HIV infection, e.g., through cytokine dysregulation (23, 24, 25), cell surface molecule modulation (26), or other mechanisms. HIV Ags coming into contact with CD4+ T cells in the absence of the appropriate costimulatory molecules or the proper cytokine milieu could induce unresponsiveness in HIV-specific CD4+ T cells (27, 28, 29). One approach in evaluating the potential contribution of unresponsiveness to the muted proliferative response of CD4+ T cells to HIV-specific Ags is to override the unresponsiveness in these cells. IL-12 (30, 31, 32) and enhanced B7-1 and B7-2-CD28 (hereafter referred to as B7-CD28) interactions (33, 34) have been shown to prevent or override anergy. In this regard, it has been demonstrated that, in vitro, IL-12 (35, 36) and IL-15 (36) can augment proliferative responses to recombinant HIV gp120 or pooled env peptides. However, p24 Ag-specific responses may be of particular importance in terms of the immune system’s ability to control HIV (9, 21, 22, 37).
CD40/CD40 ligand interactions potently up-regulate the expression of
B7-1 and B7-2 on APC (38, 39, 40) and substantially increase
the production of IL-12 and IL-15 by APC (41, 42, 43). In
addition, CD40LT induces the production of IFN- by PBMC from
HIV-infected individuals in response to mitogen by inducing the
production of IL-12 and enhancing B7-CD28 interactions (36, 44). Furthermore, IL-12 can induce the production of IFN- in
response to mitogen and non-HIV recall Ags in both HIV-infected and
uninfected persons (36, 44). We demonstrate that CD40LT
and IL-12, but not IL-15, augment PBMC and CD4+ T
cell proliferation to HIV p24 Ag in HIV-infected individuals receiving
effective HAART. In contrast, IL-12 and CD40LT have no effect on the
proliferative response to CMV Ags. We also show that CD40LT exerts its
effect through B7-CD28-dependent and IL-12- and IL-15-independent
mechanisms. Finally, CD40LT and IL-12 induce production of IFN- by
PBMC in response to p24 in HIV-infected individuals. These data suggest
the potential contribution of anergy to the lack of HIV-specific immune
responses in HIV-infected individuals and argue against clonal deletion
as the sole cause of this phenomenon. In addition, they shed light on
the possible role of CD40LT and IL-12 as possible immunomodulatory
agents for the treatment of HIV infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sixteen HIV-infected patients receiving effective HAART (groups
A–C) and seven HIV-uninfected individuals were evaluated. HIV
serostatus was determined by ELISA and Western blot. The virologic and
immunologic characteristics of the HIV-infected volunteers before and
after receiving HAART are shown in Table I
. Viral loads were determined by RT-PCR
or branched DNA assays. Patients 1–4 initiated HAART within 4 wk of
the acute primary infection syndrome and each had only two reactive
bands on Western blot (p24 and either gp120/160 or p55). Patients 5–6
began receiving HAART within 3 mo of seroconversion. Individuals in
groups B and C (patients 7–11 and 12–16, respectively) began therapy
while in the chronic stage of HIV infection; patients in group B
received HAART with a CD4+ T cell count >200
cells/mm3, while individuals in group C had more
advanced disease and began therapy with a CD4+ T
cell count <200 cells/mm3. All HIV-infected
participants had good immunologic and virologic responses to HAART
(Table I). All HIV-infected patients were positive for CMV IgG by ELISA
(data not shown).
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PBMC were isolated from whole blood by Ficoll-Hypaque centrifugation. After washing, the cells were reconstituted in RPMI with 5% pen-strep (Biofluids, Rockville, MD) and 10% human AB serum (Gemini Biological Products, Calabasas, CA) (media) at a concentration of 1.5 x 106/ml. Aliquots of 100 µl were placed in 96-well U-bottom microculture plates (Costar, Cambridge, MA) in the presence or absence of 0.5 or 5 µg/ml HIV NY5 p24, as indicated, or 5 µg/ml IIIB gp120 (Applied Biosystems, Rockville, MD; Protein Sciences, Meridian, CT; and Austral Biologicals, San Ramon, CA), 5 µg/ml CMV lysate (BioWhittaker, Walkersville, MD), 2 µg/ml CD40LT, which has been described (44) (kind gift of Immunex, Seattle, WA), 5 ng/ml IL-12 (PeproTech, Rocky Hill, NJ), 10 ng/ml IL-15 (PeproTech), 20 µg/ml rhCTLA-4 (R&D Systems, Minneapolis, MN), 20 µg/ml mAbs to IL-12 (R&D Systems), or 20 µg/ml mAbs to IL-15 (R&D Systems), as indicated. In some experiments, CD8+ and CD4+ cells were depleted with Dynal beads (Oslo, Norway), per manufacturer’s instructions. Cultures were incubated at 37°C. After 3 or 5 days, as indicated, cultures were pulsed with 1 µCi of [3H]thymidine and were harvested after 18 h. Results represent the mean of duplicate wells. The SEM was <10% in all experiments.
Measurement of cytokine production
Before the addition of [3H]thymidine,
supernatants were harvested from the 96-well plates. IFN- was
measured by commercial ELISA calorimetric kit (R&D Systems; lower limit
of detection, 3 pg/ml), according to manufacturer’s instructions.
Results represent the mean of duplicate wells. The SEM was <10% for
all experiments.
Statistics
The geometric mean was used for all proliferation assay and
stimulation index (SI) calculations. Significance for proliferation
assays was determined by an SI > 3. For IFN- production, the
Wilcoxon signed rank test was used to determine whether paired
differences were significant. p values were adjusted for
multiple testing by the Bonferroni method.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We evaluated the potential for CD40LT, IL-12, and IL-15 to enhance
proliferative responses of PBMC from HIV-infected individuals to p24
Ag. Of note, 15 of 16 individuals who were receiving effective HAART,
including 4 of 4 persons who began therapy during the earliest stages
of infection with incomplete seroconversion, had relatively low level
proliferative responses to 5 µg/ml of p24 Ag (Fig. 1A, and data not shown).
However, 14 of 16 and 10 of 13 individuals had significant
increases in proliferation to p24 with the addition of CD40LT and
IL-12, respectively (Fig. 1, A and B). The SI for
CD40LT and IL-12 was similar for 11 of 13 individuals (data not shown).
Patients 11 and 14 did not have a significant increase in proliferation
to p24 with the addition of CD40LT or IL-12, and patient 1 did not have
an enhanced response with the addition of IL-12. One of these
individuals (patient 14) had a relatively high proliferative response
to p24 alone (SI = 6 for p24 alone, CD40LT, and IL-12, data not
shown). Since 7 of 13 individuals studied (patients 1, 4, 5, 8, 11, 14,
16) did not have an SI > 3 with the addition of IL-15 (Fig. 1B and data not shown), we do not consider the results to be
significant. As an internal control, we performed nine experiments on
samples from four patients at two time points within 1 mo. There was no
significant variation in results for any of the individuals studied
(data not shown).
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C).
Consistent with previous reports that the maximal time for a
proliferative response to p24 Ag in HIV-infected individuals is 5–6
days (21), there was no significant enhancement of
proliferation to p24 Ag in four individuals, including one recently
infected person, when the [3H]thymidine
incorporation assay was performed on day 3 (Fig. 1D).
Furthermore, a relatively high concentration of p24 Ag is required to
observe a significant increase in proliferation with CD40LT and IL-12
(Fig. 1E).
Finally, since IL-2 is known to overcome anergy in certain models (45), in the initial experiments we attempted to evaluate the ability of IL-2 to induce responses to p24 Ag. However, the addition of IL-2 to the cultures resulted in an enhancement of the baseline proliferation levels, making it difficult to determine whether there was a significant increase in the response to p24 Ag with IL-2 (data not shown).
Enhanced proliferation with CD40LT and IL-12 is p24 Ag specific
In addition to using p24 as the stimulating Ag, we also performed
these experiments using HIV IIIB gp120 as an Ag. None of the
individuals had an enhancement of PBMC proliferation to gp120 with
CD40LT, IL-12, or IL-15 (data not shown). This raised the possibility
that the augmented response to p24 Ag was not specific to HIV. To
address this question, we studied seven HIV-uninfected individuals.
None of the individuals had an increased proliferative response to p24
in the presence of CD40LT or IL-12 (Fig. 2A). It was also possible that
the relatively high concentration of Ag used could induce a nonspecific
enhancement of proliferation with the addition of CD40LT and IL-12 in
HIV-infected individuals. However, there was no significant increase in
responses to CMV with CD40LT, IL-12, or IL-15 in the HIV-infected
individuals with this relatively high dose of Ag (Fig. 2B).
Except for one patient (SI = 2.9), none of the HIV-uninfected or
HIV-infected individuals studied had an SI > 2 in these
experiments (data not shown).
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CD40LT could potentially augment T cell proliferative responses
through several mechanisms. CD40LT can up-regulate expression of B7-1
and B7-2 on APCs, thereby enhancing interaction with CD28 on T cells
(38, 39, 40). CD40LT also stimulates the production of IL-12
and IL-15 by APCs (41, 42, 43). Thus, it is possible that the
CD40LT could simply be an upstream means to promote IL-12 secretion and
thereby T cell proliferation. To evaluate the potential mechanisms
whereby CD40LT enhances proliferative responses, we utilized rhCTLA-4
that is known to inhibit B7-CD28 interactions (46) and
mAbs to IL-12 and IL-15 that are known to inhibit the activity of IL-12
and IL-15, respectively (36, 44). In each of the five
experiments performed, rhCTLA-4 completely abrogated the increase in
proliferation to p24 seen with CD40LT, while mAb to IL-12 and IL-15 had
no effect (Fig. 3, A and
B). Thus, in these experiments, the effect of CD40LT is
mediated through B7-CD28 interaction.
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in PBMC stimulated
with p24 Ag
IL-12 and CD40LT have been shown to induce the production of
IFN- by PBMC and CD4+ T cells in response to
mitogens and recall Ags, including HIV gp120 (36, 44).
Therefore, we evaluated the potential of CD40LT and IL-12 to enhance
secretion of IFN- in PBMC from HIV-infected individuals exposed to
p24 Ag in vitro. Both CD40LT and IL-12 significantly increased IFN-
in the supernatants derived from cultured PBMC of 11 patients (Fig. 4). Of note, 2 of 11 patients with
enhanced proliferation as a consequence of CD40LT and 1 of 10 with
enhanced proliferation as a consequence of IL-12 did not have an
increased production of IFN- (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2). This would indicate that the unresponsiveness is
specific to HIV and not a function of global anergy or cytokine
dysregulation.
It should be noted that, while the enhanced cellular proliferation to
p24 Ag in the presence of CD40LT and IL-12 is significant, as
determined by SI (Fig. 1, A and B), the levels of
proliferation are lower than those observed in response to CMV (Figs. 1A and 2B). This could be due to the fact that
p24 is a purified protein, while the CMV Ag used in this study is a
viral lysate combing many possible Ags. However, these responses are
also significantly lower than the p24 Ag responses reported for several
HIV-infected individuals who are pre-seroconversion or who are
long-term nonprogressors (21). This discrepancy could be
due to several factors. We have addressed the possibility that the
responses we have induced are nonspecific. Since no proliferative
responses were induced to p24 Ag in seven HIV-uninfected individuals
and there was no increase in the proliferative responses to 5 µg/ml
of CMV with CD40LT or IL-12 in the HIV-infected individuals, the
responses that we have induced appear to be HIV specific. Some degree
of clonal deletion may have occurred in addition to unresponsiveness,
thereby limiting the proliferative response.
It is also possible that the level of unresponsiveness of most cells is beyond the ability of CD40LT or IL-12 to overcome in vitro, as has been previously demonstrated in certain circumstances (47, 48, 49). While we and others (35, 36) have demonstrated that IL-12 augments proliferative responses to HIV-specific Ags in HIV-infected individuals, in other systems, e.g., murine acquired immune deficiency, IL-12 has been unable to overcome anergy (49). Furthermore, when IL-12 is withdrawn from tissue cultures, rescue from anergy can be ablated (48). Although differences in the Ags and systems used may explain the divergent results, the utility of IL-12 for restoring responsiveness to HIV-specific Ags in HIV-infected patients requires further study. In addition, it is conceivable that some individuals do not have the genetic machinery necessary to mount a significant response to HIV-specific Ags, including p24. Finally, the enhanced proliferation that we observed with CD40LT and IL-12 could be a consequence of minor clones being recruited by strong immune adjuvants. Thus, the clinical significance of our findings is uncertain.
In contrast to previous reports (21, 50), we did not see a
significant proliferative response to p24 Ag in patients who began
HAART in the earliest stages of infection with incomplete
seroconversion. Indeed, the only individual with a significant response
to p24 alone was patient 14, who began receiving HAART during the
chronic stage of HIV infection with a CD4+ T cell
count of 156 cells/mm3. Since the loss of
HIV-specific responses would presumably occur at different rates in
different individuals, it is possible that our patients had advanced
past the stage of those individuals who had been previously reported
(21, 46). The lack of an enhancement of a proliferative
response to CMV by IL-12 and IL-15 (Fig. 2B) contrasts with
previous reports of enhanced proliferation to common recall Ags, e.g.,
tetanus toxoid (36). This discrepancy could be due to the
high concentration of CMV Ag used in this study, as indicated by the
strong response to Ag alone; most reactive cells may be recruited by
the relatively high level of Ag used. It is also possible that CMV and
tetanus toxoid responses are inherently different in terms of
augmentation with cytokines. The lack of a proliferative response to
HIV gp120 with the addition of IL-12 is also divergent from previous
results (35, 36). This may be due to the difference in the
source of Ag. To evaluate this, we tested two additional sources of
gp120, but still failed to induce a response (data not shown). It is
also possible that differences in doses of Ags used may account for the
discrepancy.
Since T cells can proliferate without producing cytokines
(51, 52, 53), we evaluated the function of proliferating cells
by measuring the production of IFN-. CD40LT and IL-12 induced the
production of IFN- in response to p24 Ag by PBMC of HIV-infected
individuals who were receiving effective HAART (Fig. 4). Most of these
individuals had maintained a plasma viral load of <50 copies/ml for
more than 12 mo (Table I). Using intracellular cytokine assays and
HIV-specific Ags, Pitcher et al. (54) demonstrated IFN-
induction in CD4+ T cells of HIV-infected
individuals at all stages of disease when anti-CD28 Ab and
anti-CD49d were added to short-term cultures. However, this
response was no longer detectable after 24 mo of HAART
(54). In the present study, we show that IFN- can be
induced with CD40LT and IL-12 even after prolonged effective
antiretroviral therapy. As noted, three individuals with proliferative
responses to p24 Ag in the presence of CD40LT or IL-12 did not have
increased production of IFN-. The significance of the divergence
between proliferation and production of IFN- is unclear.
The CD4+ T cell proliferative response to p24 Ag
has been proposed as an important element in the control of HIV
infection. We have demonstrated that CD40LT and IL-12 can enhance the
proliferative response to p24 Ag in vitro in HIV-infected individuals
and that this proliferative response is associated with the production
of IFN- in most individuals studied. These data suggest a
contribution of HIV-specific anergy to the inability of HIV-infected
individuals to respond to p24 Ag and argue against clonal deletion as
the sole explanation of this phenomenon. They also indicate the
potential to induce responsiveness to p24 Ag in patients effectively
treated with HAART for prolonged periods of time and suggest a possible
role for CD40LT and IL-12 as immunomodulatory therapies for HIV
infection.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: HAART, highly active antiretroviral therapy; CD40LT, CD40 ligand trimer; gp, glycoprotein; rh, recombinant human; SI, stimulation index.
Received for publication January 28, 2000. Accepted for publication May 22, 2000.
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References |
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synergistically restore IL-12 production in HIV-infected patients. Eur. J. Immunol. 28:646.[Medline]
. J. Exp. Med. 179:1109. from human peripheral blood mononuclear cells in an IL-12- and/or CD28-dependent manner. J. Immunol. 160:1701. production in primary culture: responses of subjects with allergic rhinitis and normal controls. Immunology 85:373.[Medline]
is associated with elevated levels of serum antibodies to activating malaria antigens. Proc. Natl. Acad. Sci. USA 87:5484.This article has been cited by other articles:
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) and day 5
(
), the time used in all other experiments. The geometric mean of
these four experiments is shown. In these same four individuals, p24 Ag
was used in concentrations of 0.5 and 5 µg/ml (E), the
concentration used in all other experiments. The level of
[3H]thymidine incorporation for media alone was <800
cpm/µl in all experiments.
