IL-2 Supplementation Enhance In Vitro Expansion of Anti-CD3/28 Activated CD4+ T
Cells From HIV Infected Patients
Vajee Petphong1,2,*, Nattawat Onlamoon1, Kovit Pattanapanyasat1,#
1
Division of Instruments for Research, Office for Research and Development, Faculty of
Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
2
Graduate program in Immunology, Department of Immunology, Faculty of Medicine Siriraj
Hospital, Mahidol University, Bangkok, Thailand
*e-mail: petphong@hotmail.com, #e-mail: kovit.pat@mahidol.ac.th
Abstract
Human immunodeficiency virus infection results in the severe depletion of CD4+ T
lymphocytes and damaged immune system, giving rise to the emergence of opportunistic
infections and acquired immunodeficiency syndrome (AIDS). Although highly active
antiretroviral therapy have been successfully applied to improve CD4+ T cell counts and
suppress plasma viral load, the weakening of immune responses in HIV-infected patients
cannot be fully reversed. Many alternative therapeutic strategies including transfusion of antiCD3/CD28 expanded CD4+ T cells were developed to overcome these problems. It has been
shown that anti-CD3/CD28 activation can potentially increase CD4+ T cell expansion and
render these cells resistant to M-tropic isolates of HIV-1. In order to provide CD4+ T cells
for transfusion procedure, large quantities of CD4+ T cells are needed. However, culturing
method and the degree of disease severity might have some effects on CD4+ T cell expansion
ability. In this study, we determined the levels of CD4+T cell expansion in HIV infected
patients with a good outcome. The results demonstrated that the patients with high CD4
counts (more than 500 cells per microliter) showed much lower fold expansion than health
individuals when purified CD4+ T cells were activated with anti-CD3/CD28 coated beads for
3 weeks. Additionally, this study also determined whether IL-2 supplementation helps to
further enhance the expansion of anti-CD3/CD28 activated CD4+ T cells from HIV-infected
patients. The results showed that IL-2 supplementation (100 units per milliliter) efficiently
increased the levels of fold expansion of the expanded CD4+ T cells when compared with
cell cultures in the absence of IL-2. These findings show that HIV infection have a profound
impact on the expansion of purified CD4+ T cells and anti-CD3/CD28 activation
supplemented with IL-2 at suitable concentration helps to increase the levels of CD4+ T cell
expansion. This method might provide the suitable numbers of expanded CD 4+ T cells for
transfusion therapy.
Keywords: CD4+ T cells, HIV, anti-CD3/28 activation, IL-2
Introduction
Human immunodeficiency virus type 1 (HIV-1) infection causes progressive
depletion of CD4+ T lymphocytes and dysfunction of the immune system which is
accompanied by opportunistic infections and AIDS (1). Although the use of highly active
antiretroviral therapy (HAART) contributes to a substantial reduction of viral load and a
considerable enhancement of CD4+ T cell counts, long-term use of this therapy can give rise
to drug toxicity and the emergence of drug resistant viruses, further interfering with the
complete functional restoration of the immune system (2,3). Thus, in order to improve HIVinfected patients’ quality of life and to increase their survival rates, it is essential to find an
alternative therapeutic approach that can safely promote the restoration of the immune system
as well as curtailing other destructive risk factors. Recent studies showed that CD4+ T cell
stimulation by using anti-CD3/CD28 co-immobilized beads can render the activated CD4+ T
cells intrinsically resistant to macrophage tropic (M-tropic) isolates of HIV-1 (4). The stage
of viral resistance occurs as a result of the down-regulation of CCR5 expression and the
increased production of β-chemokines (MIP-1α, MIP-1β and RANTES) of the stimulated
CD4+ T cells induced by anti-CD3/CD28 costimulation (4). Moreover, anti-CD3/CD28
costimulation can provide long-term expansion of CD4+ T lymphocytes greater than other
stimuli and induce cytokine productions with Th1 phenotype such as IL-2 and IFN-γ (5).
Interestingly, anti-CD3/CD28 activated autologous CD4+ T cell transfusion was reported to
efficiently facilitate long-term control of viral load and enhance cell-mediated immune
responses, even in the absence of antiviral chemotherapy, in chronically SIV-infected rhesus
macaques (6). In addition, it was proved that adoptive immunotherapy of anti-CD3/CD28
costimulated autologous CD4+ T lymphocytes helps increase T cell receptor (TCR) diversity
and improve immune responses in HIV-infected patients (7). Indeed, CD4+ T cell counts and
CD4:CD8 ratios were increased in dose-dependent fashion after the anti-CD3/CD28
costimulated CD4+ T cells were adoptively transferred to HIV-infected patients, indicating a
constructive way to restore the immune system in these patients (8). However, most of the
cases were observed on cells derived from HIV-infected patients in the acute or
asymptomatic stage of HIV-infection and the results sometimes can be variable among
individuals. Thus, in this study, we examine whether disease progression based on CD4+ T
cell level would have any effects on the fold expansion of isolated CD4+ T cells by using
anti-CD3/CD28 coated beads and determine whether IL-2 supplementation can further
enhance cell expansion of the anti-CD3/CD28 stimulated CD4+ T cell cultures in HIVinfected patients.
Methodology
Sample collection and CD4+ T cell isolation
Five HIV-seropositive patients who had absolute CD4 count more than 500 cell/ µl
and five healthy volunteers were enrolled in this study. The protocols were approved by the
Institutional Review Board (SIRB) of the Faculty of Medicine, Siriraj Hospital, Mahidol
University and the Faculty of Allied Health Sciences, Chulalongkorn University. Written
informed consent was obtained from all subjects prior to sample obtainment. CD4+ T cells
were directly purified from whole blood by using RosetteSep® Human CD4+ T cell
enrichment cocktail (STEMCELL Technologies, Vancouver, BC, Canada).
Activation and expansion of CD4+ T cells using anti-CD3/CD28 coated beads
The enriched CD4+ T cells purified by immunorosette formation were stimulated
with anti-CD3/CD28 coated beads: Dynabeads® Human T-Activator CD3/CD28 (Invitrogen
Dynal, Oslo, Norway). In this study, 1 million cells of isolated CD4+ T cells were activated
with anti-CD3/CD28 coated beads at 1:1 bead-to-cell ratio and the cell suspension was
adjusted to have the cell density at 5x105 cells/ml by replenishing with complete medium
(RPMI1640 supplemented with 10% fetal bovine serum, L-glutamine and streptomycin)
throughout the whole experiment. Then, the cell suspension was incubated in a humidified
CO2 incubator at 37°C for 3 weeks. Initially, the stimulation of CD4+ T lymphocytes was
performed in 24-well plates with the amount of the cells at 1x106 cells on day 0. After
incubation, the cell cultures were transferred to T25 plastic tissue culture flasks on day 4, T75
culture flasks on day 7 and T125 culture flasks on day 11 in order to avoid contact inhibitions
of cell growth. During CD4+ T cell expansion, the cell cultures were replenished with a
calculated amount of fresh media on days 4, 7, 11, 14 and 17 so as to maintain the cell
density mentioned above. On day 7, the amplified CD4+ T cells were re-stimulated with the
anti-CD3/CD28 coated beads at a 1:1 bead-to-cell ratio. Cell viability and cell numbers of
expanded CD4+ T cells were observed on days 4, 7, 11, 14, 17 and 21 by trypan blue
exclusion using a hemacytometer or TC10™ Automated Cell Counter (Biorad). To study the
effects of IL-2 supplementation, the cell cultures were supplemented with IL-2 (Protein
Specialists, CA) at a concentration of 100 U/ml along with fresh media. IL-2 supplementation
was initiated on day 7 of cell culture and maintained until the end of culture period.
Phenotypic characterizations by flow cytometric analysis
Phenotypic characterizations of the purified and expanded cells were performed by
flow cytometric anslysis. The suitable concentrations of anti-CD3 conjugated with
fluorescein isothiocyanate (FITC), anti-CD45 conjugated with peridinin chlorophyll protein
(PerCP), anti-CD8 conjugated with phycoerythrin (PE), anti-CD19 PE, anti-CD4 conjugated
with allophycocyanin (APC), anti-CD16 APC and anti-CD56 APC were used for cell
staining. Samples were acquired by a BD FACSCalibur flow cytometer (BDB, San Jose, CA)
and the collected data were analyzed by FlowJo Software (Tree Star, Eugene, OR).
Statistical analysis
Collected data from the expansion of CD4+ T lymphocytes and the frequency of
lymphocyte subsets were shown as mean ± standard deviation (S.D.). The Mann-Whitney U
test was used to determine the statistical differences between each group of samples. The
statistical differences of the mean values of fold expansion between cell cultures in the
absence of IL-2 and those in the presence of IL-2 were determined by Paired Sample t-test. Pvalues < 0.05 were considered statistically significance.
Results
The fold expansion was determined based on the numbers of cells at the end of
culture period divided by the numbers of cells at the beginning of cell expansion. The results
showed that the average fold expansions on days 4, 7, 11, 14, 17 and 21 of healthy
individuals were 3.2±0.2, 12.7±2.8, 36.2±9.7, 108.3±35.7, 482.3±214.0 and 1,202.5±183.8,
respectively. The average percentages of cell viability of healthy subjects on the indicated
days were 91.9±2.8, 94.9±1.0, 86.0±9.8, 89.2±7.2, 96.2±1.2 and 95.4±3.3, respectively. For
the HIV-infected patients, the average fold expansions on days 4, 7, 11, 14, 17 and 21 were
3.2±0.6, 13.4±4.4, 36.0±13.7, 109.8±68.9, 385.3±352.1 and 878.4±334.6, respectively. The
average percentages of cell viability of HIV-infected patients on the days mentioned were
88.3±6.5, 90.2±1.3, 82.7±7.5, 93.7±2.8, 94.9±2.3 and 92.9±1.6, respectively. As shown in
figure 1, these data demonstrated that healthy donors showed the higher fold expansion of
CD4+ T cells (1,202.5±183.8) compared with HIV-infected patients (p = 0.076) after cell
expansion for 21 days.
Fold expansion
P = 0.076
1400.0
1200.0
1000.0
800.0
600.0
400.0
200.0
0.0
HIV infected
patients
Healthy
individuals
Figure 1. The average fold expansions of anti-CD3/28 expanded CD4+ T cells from HIV-infected patients and
healthy individuals on the final day of cell culture.
Phenotypic characterizations of anti-CD3/CD28 expanded cells from HIV-infected
patients and healthy individuals were determined. The results showed that the expanded cells
from both healthy individuals and HIV-infected patients showed high frequencies of CD4+ T
cells after a 3-week culture period of cell expansion. The average percentages of CD4+ T
cells among lymphocytes on days 14 and 21 of healthy individuals were 94.3±3.1% and
96.6±2.3% whereas the average percentages of CD4+ T cells among lymphocytes on days 14
and 21 of HIV-infected patients were 91.4±2.1% and 95.7±1.0%.
An attempt to increase the fold expansion of CD4+ T cells from HIV-infected patients
using IL-2 supplementation was determined. The isolated CD4+ T cells obtained from HIVinfected patients were stimulated with anti-CD3/CD28 coated beads (1:1 bead-to-cell ratio)
and expanded either in the absence or in the presence of IL-2 supplementation at the
concentration of 100 U/ml. The calculated amounts of the cytokine were added to the cell
cultures on days 7, 11, 14 and 17. In the absence of IL-2 supplementation, the fold expansion
of CD4+ T cells in HIV-infected patients on the indicated days were 3.7±0.1, 11.7±2.4,
38.3±12.6, 104.8±32.5, 244.7±51.6 and 584.4±37.3, respectively. Interestingly, in the
presence of IL-2 supplementation, the fold expansion of CD4+ T cells in HIV-infected
patients on the indicated days were 3.4±0.7, 12.6±4.1, 36.5±2.0, 118.7±8.7, 387.4±62.9 and
1335.5±173.2, respectively. It is noticeable that IL-2 supplementation enhanced the
expansion of the cells in both healthy individuals and HIV-infected patients as shown in table
1.
Table 1. Fold expansions of anti-CD3/28 expanded CD4+ T cells from HIV-infected patients and healthy
individuals with IL-2 supplementation. Data were obtained from 3 subjects for each group.
Fold expansion
Subject
HIV-infected
patients
Healthy individuals
Day
IL-2 supplementation
Donor
1
Donor
2
Donor
3
Mean ± SD
0
1.0
1.0
1.0
1.0 ± 0.0
4
4.1
2.7
3.6
3.4 ± 0.7
7
14.8
7.8
15.1
12.6 ± 4.1
11
38.6
36.2
34.7
36.5 ± 2.0
14
110.4
118.0
127.7
118.7 ± 8.7
17
389.8
323.3
449.1
387.4 ± 62.9
21
1,493.9
1,150.6
1,361.9
1,335.5 ± 173.2
0
1.0
1.0
1.0
1.0 ± 0.0
4
3.0
2.8
3.8
3.2 ± 0.5
7
10.9
12.8
12.0
11.9 ± 1.0
11
43.6
42.2
53.8
46.5 ± 6.3
14
110.3
195.0
209.3
171.5 ± 53.5
17
443.3
789.2
694.0
642.1 ± 178.7
21
1,836.0
2,609.0
2,340.0
2,261.7 ± 392.4
Discussion and Conclusion
Many studies have been exploring the influences of cell transfusion therapy using
anti-CD3/CD28 activated CD4+ T lymphocytes in both HIV-infected nonhuman primate
models and HIV-infected patients and the results showed that this therapeutic treatment can
help to boost tremendous immune responses against HIV-1 and competently control viral
replication (9-11). However, although many findings have proved that stimulation by using
anti-CD3/CD28 coated beads can substantially augment cellular proliferation of CD4+ T
lymphocytes and render the cells refractory to M-tropic isolates of HIV-1, the impact of HIV
infection to the expansion ability of CD4+ T cells activated by anti-CD3/CD28 coated beads
had never been investigated.
The expansion ability can vary among individuals since HIV-1 cannot infect
lymphocytes from different subjects at an equivalent capacity which raising the question
about the association between the effects of different rates of disease progression and the
stage of viral resistance of lymphocytes to HIV infection in vitro (12, 13). In this study, we
examined the effects of HIV infection on the fold expansion and cell viability of CD4+ T
cells using anti-CD3/CD28 coated beads. The results showed that although HIV-infected
patients had high CD4 counts (>500 cells/µl), they exhibited much lower fold expansion
when compared to health individuals. We provide evidence that the stages of disease
progression may have a crucial impact on the fold expansion and survival rates of CD4+ T
cells of HIV-infected subjects.
Furthermore, we determined an optimal IL-2 concentration for the expansion of
purified CD4+ T cells and investigated whether IL-2 supplementation at the optimal
concentration will increase fold expansion of anti-CD3/CD28 expanded CD4+ T cells in
HIV-infected patients. Our data show that the addition of exogenous IL-2 at the concentration
of 100 U/ml can greatly restore proliferative response in CD4+ T cells from HIV-infected
subjects. Cultures supplemented with exogenous IL-2 showed higher levels of fold expansion
than those without IL-2. However, the fold expansion in HIV-infected patients still lower
than those observed in healthy donors.
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Acknowledgements: Siriraj Graduate Thesis Scholarship