Qualitative Immune Modulation by Interleukin-2 (IL-2)
Adjuvant Therapy in Immunological Non Responder HIV-
Francesca Sabbatini1*, Alessandra Bandera1, Giulio Ferrario2, Daria Trabattoni3, Giulia Marchetti4, Fabio
Franzetti2, Mario Clerici5, Andrea Gori1
1Division of Infectious Diseases, Department of Internal Medicine, San Gerardo Hospital, University of Milan-Bicocca, Monza, Italy, 2Department of Clinical Sciences,
Infectious Diseases Section, Luigi Sacco Hospital, University of Milan, Milan, Italy, 3Department of Preclinical Sciences, Chair of Immunology, LITA VIALBA, University of
Milan, Milan, Italy, 4Department of Medicine, Surgery and Dentistry, Clinic of Infectious Diseases, San Paolo Hospital, University of Milan, Milan, Italy, 5Chair of
Immunology, Milan University Medical School and Don C. Gnocchi Foundation IRCCS, Milan, Italy
Background: Treatment of HIV-infected patients with interleukin-2 (IL-2) produces significant increases in CD4 T cell counts;
however an associated qualitative improvement in cells function has yet to be conclusively demonstrated. By measuring
mycobacterial killing activity, we evaluated IL-2-mediated functional immune enhancement ex vivo in immunological non-
Methods and Findings: PBMC from 12 immunological non-responders (INRs) (CD4+,200/ml, HIV-RNA,50 cp/ml) on
combination antiretroviral treatment (cART) were collected at baseline, and after 3 IL-2 cycles. Eight INRs receiving only
cART were studied as controls. After 21 days of PBMC incubation with a virulent M. avium suspension, counts of residual
colony forming units (CFUs) and concentrations of TNF-a, IL-10 and IFN-c were determined. In IL-2 treated patients, a
significant reduction in mean residual CFUs of PBMC cultures was observed (p,0.01). Moreover, following IL-2 treatment,
significant increases in PBMC’s IFNc production (p=0.02) and substantial reductions in IL-10 levels were observed.
Conclusions: IL-2 therapy restores the ability of the lympho-monocyte system in eliciting an effective response against
mycobacterial infections. Our data indicate the possibility of a clinical role held by IL-2 in enhancing the immune function of
subjects unable to achieve immune competence through cART alone.
Citation: Sabbatini F, Bandera A, Ferrario G, Trabattoni D, Marchetti G, et al. (2010) Qualitative Immune Modulation by Interleukin-2 (IL-2) Adjuvant Therapy in
Immunological Non Responder HIV-Infected Patients. PLoS ONE 5(11): e14119. doi:10.1371/journal.pone.0014119
Editor: Lishomwa C. Ndhlovu, University of California San Francisco, United States of America
Received July 2, 2010; Accepted November 2, 2010; Published November 29, 2010
Copyright: ? 2010 Sabbatini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The present study was funded by a grant from the Ermenegildo Zegna Foundation, Italy, and Angelantoni Heraeus Scientific Instruments (A.H.S.I.)
S.p.A., Milan, Italy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Since its introduction into clinical practice, combination
antiretroviral treatment (cART) has dramatically changed the
course of HIV natural history, assuring optimal inhibition of HIV
replication and leading to effective immune recovery . Despite
these advances, up to 40% of patients beginning cART with
CD4+,50 cell/mL fail to achieve satisfactory CD4+ cells count
levels, even after years of virologically effective antiretroviral
treatment . Among such individuals, Immunological Non Responders
(INRs) stands out as CD4+ T cells in these patients remain
consistently below 200 cells/mL [3,4]. The underlying mechanism
for this lack of immune response remains undiscovered. Persistent
low CD4+ T cells count is a strong predictor of disease
progression, death and development of non AIDS-related clinical
events , and finding successful strategies for enhancing immune
response in these patients is therefore essential. Given their
particular problems in achieving satisfactory CD4+ levels despite
cART, INRs are an intriguing target for immune-based
approaches. Treatment of INRs with intermittent cycles of
interleukin-2 (IL-2) was reported to result in significant increases
in CD4+ T cells count . By inducing a polyclonal expansion of
CD4+ cells mainly characterized by a naı ¨ve or central-memory
phenotype , IL-2 greatly enhances the proliferation and
function of T-lymphocytes and natural killer cells and promotes
the production and release of other cytokines . IL-2 is
particularly active on CD4+ T cells, both in enhancing
proliferation of existing T cells and in temporarily boosting
neothymopoiesis . However, extended analysis of the qualitative
features of these newly recovered cells is required in order to define
the true functional advantages of IL-2 therapy. Recently, two
randomized prospective studies, SILCAAT and ESPRIT ,
failed to demonstrate the clinical benefit of IL-2 adjuvant
treatment. However, these trials were based on patients whose
median CD4+ cells count were at least 200 cells per ml, compared
to INRs who were the object of our own study. The aim of our
study was to determine whether CD4+ cells gain induced by IL-2
in a population of INRs was also associated with qualitative and
PLoS ONE | www.plosone.org1November 2010 | Volume 5 | Issue 11 | e14119
functional improvement of the immune performance. As a model
for evaluating qualitative immune function, we measured killing
activity on mycobacteria in peripheral blood mononuclear cells
(PBMC) ex-vivo, from a group of INRs treated with intermittent
cycles of IL-2. Reduced mycobacterial growth and enhanced
production of certain cytokines involved in antigen specific
response following IL-2 administration were considered to be
signs of enhanced immune response.
Samples analyzed in our study derived from patients enrolled in
an open-label randomized trial whose main results were published
in 2002 . HIV-infected immunological non responders enrolled
in our study received effective antiretroviral treatment alone or in
association with IL-2. The study was approved by institutional
ethic committee of Institute of Infectious Diseases, Luigi Sacco,
Milan. All individuals were enrolled by the Department of
Infectious Diseases of the Luigi Sacco Hospital in Milan. Patients
participating in this study gave written informed consent according
to the Declaration of Helsinki.
Study design and population
This study has been conducted selecting patients from 22
subjects enrolled in an open-label randomized Institutional
Review Board-approved trial of IL-2 immunotherapy in associa-
tion with cART versus cART alone, published by Marchetti et al
. The trial anticipated the ability of IL-2 adjuvant therapy of
rescuing the CD4+ T cells compartment when used at low dose for
a prolonged period. Twenty HIV-infected INRs patients were
borrowed from the main trial and their samples analyzed. All
subjects shared HIV RNA,50 copies/ml and CD4+ T cells count
consistently ,200 cells/mL after at least 6 months of stable cART.
Twelve patients received recombinant human IL-2 (Proleukin;
Chiron) administered at a dosage of 36106IU daily as a single
subcutaneous injection at days 1–5 and 8–12 of a 4 -week cycle,
for a total of 3 cycles (overall, 10 weeks’ duration), in addition to
ongoing antiretroviral treatment (Fig. 1). The other 8 patients
received cART alone. Persons who had previously been treated
with IL-2 were excluded. Patients were evaluated at baseline and
after three cycles of IL-2 and followed over 48 weeks. Peripheral
blood mononuclear cells (PBMC) were obtained at baseline prior
to IL-2 treatment (day 0: T0) and after 3 cycles of IL-2 therapy
(day 75: T1) in IL-2 treated patients and at enrollment (day 0: T0)
and after 75 days (day 75: T1) in cART-treated controls.
Patients had an overall median CD4+ T cell count nadir of 53
cells/mL. Nadir were similar between the groups: median CD4+
cells number in treated patients was 54 cells (IQR, 50.5–80 cells/
mL); median CD4+ cells number in controls was 38.5 (IQR,
18.75–54 cells/mL); P=0.26. All patients received a combination
antiretroviral regimen including either 2 nucleoside reverse-
transcriptase inhibitors and 1 protease inhibitor or 1 non-
nucleoside reverse-transcriptase inhibitor for a median time of
322 days (10.7 months) before enrolment; no differences were seen
in length of antiretroviral treatment between IL-2 treated patients
(median 322 days; IQR, 247–394 days) and controls, (median
310.5 days; IQR, 271–432.7; P=0.72). Patients demonstrated
sustained suppression of viral replication for at least 6 months
before enrolment (HIV-1 RNA levels of ,50 copies/mL in 3
consecutive determinations), and had CD4+ T cell counts
consistently below the threshold of 200 cells/mL during cART.
There were no baseline differences between the 2 groups with
respect to age, epidemiologic data, CD4+ T cell counts, HIV RNA
load, HIV-related parameters, or cART regimen at enrolment
(Table 1). None of the patients reported opportunistic infections
up to 1 year before study initiation. No patients had previous
episodes of MAC infections, and therefore none were taking MAC
primary or secondary prophylaxis.
Mycobacterium avium and inoculation of PBMC cultures
A virulent strain of M. avium was isolated and identified from a
clinical sample of a patient affected by Mycobacterium avium
disseminated infection [11,12]. For inoculation of PBMC cultures,
an M. avium stock aliquot was thawed and suspended in isotonic
saline. The M. avium suspension was homogenized and sonicated
in a sonicating waterbath for 3 min at room temperature to
disrupt mycobacterial clumps. Mycobacterial concentration was
determined through optical density (OD) and adjusted to 36107/
ml. The M. avium suspension was serially diluted 10-fold and
triplicate 10-ml spots of the dilutions were plated on agar medium
(Middlebrook 7H10 plus 10% OADC, Becton Dickinson, USA) to
Figure 1. Schedule of IL-2 administration.
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verify the number of organisms per milliliter of the inoculation
suspension . The mycobacterial suspension was utilized to
inoculate 1- and 3-6106cells/well PBMC cultures, adding 33- and
100-ml of the suspension/well respectively, at a ratio of 1 organism
per cell. For each donor, PBMC cultures were prepared in order
to allow for the following tests in duplicate, at T0 and T1: PBMC
at 1- and 3-6106cells/well (2 ml culture medium volume); M.
avium-inoculated PBMC at 1- and 3-6106cells/well (2 ml culture
medium volume), mycobacteria/cells ratio being 1:1. Cultures
were incubated at 37uC and 5% CO2, for as long as 21 days, the
mean time for mycobacterial replication. Cell count and viability
were assessed by trypan blue exclusion dye assay at the conclusion
of tests, using control cultures; cell typing of residual cells was also
performed by flow cytometry (murine mAb anti-human CD14 by
Coulter Electronics Inc., Miami Lakes, FL, USA and Epics Elite
cytometer by Coulter Electronics) at the conclusion of tests, using
Evaluation of mycobacterial growth by CFU counting
After 21-days incubation, M. avium-infected macrophages were
lysed by SDS (final concentration 0.1%) for 15 min at room
temperature. The SDS was neutralized by adding an equal volume
of 20% bovine serum albumin to each well and cell lysates were
flushed repeatedly with an automatic pipette. The cell lysates were
harvested, sonicated for 20 seconds, serially diluted 10-fold, and
plated on Middlebrook 7H10 plus OADC agar in 6-cm petri
dishes (three 10-ml spots were plated per dilution). The agar plates
were incubated for 28 days at 37uC/5% CO2and individual M.
avium colonies were enumerated with an inverted microscope after
14 and 28 days of incubation.
Cytokine Elisa assays
Because of their ascertain role in immune activation against
mycobateria, we evaluated supernatant concentration of TNF-a,
IFN-c and IL-10 from M. avium- infected PBMC cultures.
Supernatants (500mL) were harvested and replaced with fresh
culture medium at day 0 (immediately before M. avium
inoculation), at day 7, day 14 and at day 21 (immediately before
lysing of infected cultures). Supernatant samples were stored at
220uC. Cytokine concentrations was performed as per manufac-
turer’s instructions using commercial kits (Endogen Human Elisa,
Endogen, Inc., Woburn, MA, USA; detection limit of 5 pg/ml).
PBMC cultures from different donors varied as to M. avium
growth. For this reason, number of surviving bacteria were
calculated both as absolute value and as percentage variation.
Results were expressed as median number of colony-forming-units
(CFU) and Inter Quartile Range (IQR) per milliliter of lysate of
each experimental condition. Cytokine concentrations were
expressed as median pg value and IQR per milliliter of each
experimental condition. Mann-Whitney test and Student’s paired
and unpaired t-test were performed in order to determine the
statistical significance of differences in M. avium growth and
cytokines concentrations. Correlation analysis was made using a
Effects of IL-2 treatment on M. avium growth
M. avium was present in all PBMC cultures and residual CFUs
produced colonies primarily of flat, transparent type. Analysis of 1-
6106cells/well cultures in IL-2 treated patients resulted in a
median number of CFU/mL of 1450 (IQR, 1245–2035) at T0 and
of 420 (IQR, 135–1545) at T1 (P=0.008). Control patients
displayed a median CFU number of 1725 (IQR, 1247–2202.5) at
T0 and of 1590 (IQR, 1025–2020) at T1 (P=0.16). In the second
experiment, using 3-6106cells/well, the median number of CFU
was 3340 (IQR, 3167.5–4800) at T0 and 670 (IQR, 260–1782.5)
at T1 in IL-treated patients, (P=0.001), while in PBMC from
patients receiving only cART, median CFU number was 3220
(IQR, 2105–4552.5) at T0 and 3015 (IQR, 2037.5–4345) at T1
Table 1. Baseline characteristics of patients enrolled in the study.
CharacteristicIL-2 treated group (Nu u12)Control group (Nu u8)
Male sex (%)8 (66.6%) 6 (75%)
Median age [IQR]38 [33.5–41]42.5 [39.25–47.25]
Risk factors for HIV infection
HIV RNA, ,50 copies (%)100100
CD4+ + cell count nadir (per mm3)
Median (IQR) 54 [50.5–80] 38.5 [18.75–54]
Nu u of opportunistic infections at baseline (%)00
Patients on PCP profilaxis (%)100100
Patients on MAC profilaxis (%)00
Current antiretroviral treatment (%)
Median number of days spent on antiretroviral treatment
prior to enrollment [IQR]
322 [247–394]310.5 [271–432.7]
IQR Inter Quartile Range; PI: Protease Inhibitors; NRTI: Nucleoside Reverse Transcriptase Inhibitors; NNRTI: Non-Nucleoside Reverse Transcriptase Inhibitors.
IL-2 in HIV-Infected INR
PLoS ONE | www.plosone.org3 November 2010 | Volume 5 | Issue 11 | e14119
(P=0.106); (Fig. 2). This significant reduction in mycobacterial
growth suggests an improved killing activity of PBMC after IL-2
Effects of IL-2 treatment on cytokines production
Supernatant concentrations of cytokines IL-10, TNF-a and
IFN-c were determined by ELISA assay on samples harvested at
days 0, 7, 14 and 21 from inoculation. Cytokines release by PBMC
with culture medium alone was virtually undetectable in every
donor group at each determination performed before day 21 (days
0, 7, 14; data not shown). No significant differences were observed
in TNF-a concentrations at T1, when comparing treated patients
to controls. Likewise, at day 21, non significant variation in TNF-a
concentrations from T0 to T1 was observed, in both IL-2 treated
patients (T0 median value 111 pg/ml; IQR, 59.5–165.8; T1
median value 116 pg/ml; IQR, 83–307.9; P=0.07), and controls
(T0 median value 94 pg/ml; IQR 78.5–117.8; T1 median value
191 pg/ml; IQR, 159.8–235.4; P=0.06) (Fig. 3 A,B). IL-2 treated
patients presented a reduction in IL-10 concentrations (T0 median
value 79 pg/ml; IQR, 66–123.2; T1 median value 8 pg/ml; IQR,
65.4–110), whereas no changes were shown in controls (T0
median value 121 pg/ml; IQR 107.1–161.9; T1 median value
119 pg/ml; IQR, 81.2–132.3) resulting in significantly different
IL-10 levels at T1 between the two groups (P=0.03; Fig. 3 C,D).
Finally, IFN-c production substantially increased in IL-2 treated
patients between T0 and T1 (T0 median value 18 pg/ml; IQR,
31.2–67; T1 median value 87 pg/ml; IQR, 38.6–248; P=0.02)
whereas no significant changes were observed in controls between
the two time points (T0 median value 85 pg/ml; IQR, 38.5–
124.82; T1 median value 163 pg/ml; IQR, 116.23–228.6;
P=0.13) (Fig. 3 E,F).
Correlation between mycobaterial killing and cytokines
To evaluate if a direct link between the effects of IL-2 treatment
on cytokines production and mycobacterial killing activity exists,
we investigated if in IL-2 treated patients and controls MAC
growth correlates with levels of IL-10, TNF-a and IFN-c. In IL-2
treated patients a significant negative correlation was seen between
Mycobaterium avium CFU and TNF-a concentration. (r=20.89,
P=0.02), thus revealing a possible connection between the
production of this cytokine and the improved killing activity.
Moreover, in IL-2 treated patients IL-10 concentrations were
inversely associated with TNF- a levels (r=20.89, P=0.02),
whether a direct correlation was found between IFN- c and TNF-
a production (r=0.97, P=0.001).
We explored the role of IL-2 treatment in improving the
functional ability of lympho-monocytes in deeply immunocom-
promised patients with HIV infection. Accordingly to previous
Figure 2. Mycobacterial growth (CFU: Colony Forming Unit) before (T0) and after (T1) IL-2 adjuvant treatment (A,C) compared to
cART treatment alone in control group (B,D). CFU growth has been tested in different aliquots of PBMC cultures of 16106cells/well (A,B) and
36106cells/well (C,D). In IL-2 treated patients a significant reduction in mean residual mycobacterial growth was observed in both experimental
conditions of PBMC cultures (P,0.01). Significant results are emphasized by square brackets with respective P values.
IL-2 in HIV-Infected INR
PLoS ONE | www.plosone.org4 November 2010 | Volume 5 | Issue 11 | e14119
findings of the main trial , our data show that IL-2 adjuvant
treatment is effective not only in raising CD4+ cells in number, but
also in enhancing the specific immune system functional
properties. In particular results herein show that IL-2 is associated
with increased mycobacterial killing possibly due to inducing
cytokine release and synthesis. Up to 44% of patients starting
cART with very low CD4+ cells number are unable to reach a
CD4 cell count .500 cells/m despite years of virological
suppression . Several studies demonstrate that INRs, when
compared to patients who fully respond to antiretrovirals , are
prone to a higher incidence of AIDS defining illnesses or other
opportunistic events typical of clinical progression [15,16].
Effective strategies aimed at achieving improved immunological
status for these patients  are required, and several immuno-
modulant approaches, including cytokines able to stimulate cell-
mediated immunity, have been attempted [18,19]. Previous
studies have demonstrated that IL-2 effects a significant, sustained
and selective rise in CD4+ -T cells number by stimulating CD4+
cells neothymic synthesis, redistribution of central and effector
memory T cells and improving the functionality of such cells. This
suggests a possible role of this cytokine in selectively reconstituting
T helper activity to neoantigens [20,11]. Nevertheless, results of
two randomized prospective studies in HIV positive patients with
CD4+.200 cell/mL , recently failed to demonstrate clinical
benefits in IL-2 treated patients compared to cART alone; in fact,
IL-2 treated subjects and controls experienced a similar rate of
opportunistic diseases and deaths. However, INRs population was
not specifically targeted in either of these trials. On the contrary,
both studies involved patients where median CD4+ cells count was
at least 202 cells per mm3, significantly higher than in our study
(mean CD4 cells nadir: 56,7 cells/mm3). Notably, the number of
AIDS and non-AIDS related events is significantly lower in
Figure 3. Production of TNF-a (A,B), IL-10 (C,D) and IFN-c (E,F) in PBMC cultures before (T0) and after (T1) IL-2 adjuvant treatment
(A,C,E) compared to cART treatment alone in control group (B,D,F). Following IL-2 treatment significant increases in PBMC’s IFN-c
production (P=0.02), and substantial reduction in IL-10 levels were observed resulting in significant difference between IL-2 patients and controls at
T1 (P=0.03). Significant result in IL-2 treated patients are emphasized by black arrows indicating the compared groups with respective P value.
IL-2 in HIV-Infected INR
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persons with better immunological status, such as the patients
enrolled in SILCAAT and ESPRIT. Therefore, while these trials
indeed confirm the ability of IL-2 to induce sustained quantitative
gains in CD4+ cells, they fail to find a clinical advantage to the
growth of these new cells, leaving unresolved the question of their
properties. Nevertheless, SILCAAT and ESPRIT did provide a
promising hypothesis: that IL-2 may enhance proliferation of T
cells pools which are different from those expanded by
antiretroviral therapy alone and characterized by naı ¨ve and
central memory phenotypes. We attempted to demonstrate if
indeed this higher number of cells was accompanied by a
qualitative improvement in immune function. To this end, we
tested the quality of the immune response by evaluating the ex vivo
mycobacterial killing capacity of IL-2 induced lympho-monocyte
cells in a population of INRs treated with IL-2. Previously, several
authors [21,22] have shown that Mycobacterium avium killing is
defective in cells of HIV-infected patients . Herein we show
that such defect can be corrected with IL-2 administration. In
particular IL-2 appears to restore the ability of the lympho-
monocyte system in mounting an effective defence against
mycobacterial infections enhancing killing activity. Interestingly,
the improved killing activity parallels an increased release of
specific cytokines, whose production is altered during HIV
infection. Following IL-2 treatment we detected decreased levels
of IL-10, a macrophage-suppressive cytokine. Furthermore, IL-2
administration in our study resulted in higher concentrations of
IFN-c, a key mediator of bactericidal activity of macrophages.
Inhibition of the release of cytokines such as IFN-c is in fact one of
the classic IL-10 functions . Our data suggest a potential link
between macrophage activation and IL-2-induced modulation of
cytokines release. These results reflect the complexity of the
immune response to mycobacterial infections induced by IL-2, in
which Th1 cytokines such as IFN- c function as activators of
macrophages, thereby promoting intracellular killing. In the
context of mycobacterial infection, IL-10 reduction enhances
actions of stimulatory cytokines contributing to host cells defence
. Our findings indicates that in vivo IL-2 therapy results in a
beneficial immune modulation in individuals in whom cART does
not significantly improve CD4 counts. Because of the higher risk of
developing opportunistic infections or AIDS and non AIDS-
related events, patients with very low immunologic competences
may benefit from IL-2 adjuvant treatment more than patients who
fully respond to antiretrovirals. Careful identification of target
patients could offer a supplementary opportunity for immunolog-
ical improvement in particular kinds of HIV- infected subjects.
We thank Michael Alan Rosen for a critical reading of the manuscript and
valuable grammatical advice. We particularly thank all the patients
participating in the study and the staff of the divisions of Infectious Diseases
of ‘‘Luigi Sacco’’ Hospital, who cared for the patients. Findings of this
paper have partly been presented in the 16thConference on Retroviruses
and Opportunistic Infections, February 8–11, 2009, Montreal, Canada
Conceived and designed the experiments: GM FF MC AG. Performed the
experiments: GF DT MC. Analyzed the data: FS AB. Wrote the paper: FS
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PLoS ONE | www.plosone.org6 November 2010 | Volume 5 | Issue 11 | e14119