| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Medicine, University of California, San Francisco, CA 94143
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Optimal HIV production in CD4+ T cells requires not only the engagement of the TCR/CD3 complex but also a costimulatory signal provided by the CD28 molecule on the cell surface (7). Blocking the CD28 ligands, CD80 and CD86, during stimulation of PBMC reduces the ability of these cells to support HIV replication (8). Moreover, CD28 costimulation using anti-CD28 Abs or following coculture with cell lines expressing CD80 and CD86 enhances the level of HIV production when compared with cells stimulated with anti-CD3 Abs alone (9).
In an apparent contradiction to the above observations, recent studies have demonstrated that exposure to anti-CD3 and CD28 Abs immobilized on Sepharose beads appears to induce CD4+ cells to resist productive infection by strains of HIV that are able to infect macrophages (M-tropic)4 but not T cell lines (TCL-tropic) (10). This arrest in virus production is not only apparent by a decrease in virus particles present in the culture fluids but also by a reduction in proviral DNA in acutely and naturally infected cells (10, 11). The differences in the outcomes of these recent studies vs the earlier ones (9) showing enhanced HIV replication following CD28 costimulation have been attributed to the use of soluble instead of immobilized anti-CD28 Ab during stimulation (10). Following CD28 costimulation, an increased production of ß-chemokines (11) and/or decreased expression of CCR5 chemokine receptor (12) have been suggested to be involved in blocking HIV infection of CD4+ cells. Both mechanisms can prevent entry of M-tropic viruses that use CCR5 as a coreceptor (reviewed in 13 . Because CD28 costimulation appears to render CD4+ cells resistant to infection with HIV, its potential therapeutic use for ex vivo expansion of CD4+ cells from HIV-infected individuals followed by reconstitution has been proposed (10, 14).
In previous studies, we demonstrated that CD28 costimulation of CD8+ cells increased their capacity to suppress HIV replication (15). To determine whether triggering the CD28 molecule during stimulation could be used in the treatment of HIV-infected individuals, we further evaluated what effect CD28 costimulation would have on HIV infection of CD4+ cells. Our present findings demonstrate that CD28 costimulation, using anti-CD3 and CD28 Ab coupled to beads, can lead to enhanced or reduced HIV production in CD4+ cells depending on whether the cells are continuously exposed to the Ab beads during culture.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Peripheral blood was collected by venipuncture in Vacutainer tubes containing sodium heparin (Becton Dickinson, Rutherford, NJ). Blood from HIV seronegative donors was provided by The Blood Centers of the Pacific (San Francisco, CA) or came from randomly selected healthy individuals employed at the University of California, San Francisco (UCSF). The two HIV-infected individuals used in our study were volunteers with CD4+ cell counts of 682 and 164 cells/µl, respectively, and their percentages of CD4+ cells were 25 and 12, respectively. Both subjects were asymptomatic, had viral loads <500 viral RNA copies/ml at the time of the study, and were taking AZT and 3TC (Glaxo Wellcome Pharmaceuticals, Research Triangle Park, NC. The subject with 164 CD4+ cells/µl was also taking Viracept (Agouron Pharmaceuticals La Jolla, CA). The biologic phenotype of the viruses present in the blood of the infected individuals was determined by the MT2 assay (16). The studies received the approval of the Committee on Human Research, UCSF.
Isolation of PBMC and their cell subsets
PBMC were obtained by Ficoll-Hypaque (Sigma, St. Louis, MO) gradient centrifugation (17). The CD4+ cells were isolated from the PBMC by positive selection using magnetic beads bearing anti-CD4 mAb (Dynal, Lake Success, NY) (18). Beads were removed from the cells by Detach-a-bead (Dynal) according to the manufacturer’s instructions. Purity of the cells obtained by the immunomagnetic bead isolation procedure was >95% CD4+, <1% CD8+, <1% CD19+, <1% CD56+, and <1% CD14+ as determined by flow cytometry (FACSort, Becton Dickinson) (19).
Culture medium, cytokines, and reagents
RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented
with 10% heat-inactivated (56°C, 30 min) FBS (Gemini Bio-Products,
Calabasas, CA), 2 mM glutamine (BioWhittaker), and 100 U/ml penicillin,
and 100 µg/ml streptomycin (BioWhittaker) was used as the culture
medium (complete medium). Stimulation of purified CD4+
cells (3 x 106) for use in the acute infection was
done in the presence of 1 ml of the complete medium containing 100 U/ml
of recombinant human (rh) IL-2 (gift from Glaxo-Wellcome) over 3 days.
The mouse anti-human CD3 mAb (clone X35; Immunotech, Bar Harbor,
ME) used to stimulate the CD4+ cells was coupled to
tosyl-activated magnetic Sepharose beads according to the
manufacturer’s instructions (Dynal). The optimal anti-CD3 Ab
bead:CD4+ cell ratio used in our study was 2:1. This ratio
was determined by the maximal incorporation of tritiated thymidine by
CD4+ cells incubated for 3 days in the presence of various
numbers of beads compared with cells cultured in the absence of the
beads in a final volume of 0.2 ml of complete medium (Fig. 1
A). In experiments evaluating
costimulation of CD4+ cells, anti-CD28 Ab (clone
CD28.2; Immunotech) was added together with equal amounts of
anti-CD3 Ab to tosyl-activated beads and used at a optimal
bead-to-cell ratio of 2:1 (Fig. 1B). The CD80Ig and CD86Ig
fusion proteins used to costimulate CD4+ cells (provided as
a gift by Philip Morton, Monsanto, St. Louis, MO) were mixed with an
equal amount of anti-CD3 Ab before coupling them to magnetic rat
anti-mouse IgG2a (Fc) beads according to the manufacturer’s
instructions (Dynal). These beads were used at an optimal bead-to-cell
ratio of 2:1 (data not shown).
|
Following stimulation of the CD4+ cells, the
magnetic beads were removed and the cells were washed three times in
calcium- and magnesium-free HBSS (CMF-HBSS; BioWhittaker). The
stimulated CD4+ cells were then treated with polybrene (2
µg/ml for 30 min; Sigma) and infected with 100 x 50% tissue
culture infectious dose (TCID50) of HIV-1SF33,
HIV-1SF162HIV-1SF128A, or HIV-1BaL
for 1 h. The HIV-1SF33 isolate is a highly cytopathic,
TCL-tropic, and syncytium-inducing (SI) virus (20). The other isolates
are nonsyncytium-inducing (NSI) M-tropic viruses (21, 22). All viruses
were cultured only in PBMC and not T cell lines. The ß-chemokines,
RANTES, MIP-1
, and MIP-1ß, have been shown to block infection of
the M-tropic but not the TCL-tropic viruses (23). The infected cells
were washed three times with CMF-HBSS, resuspended in complete medium
containing 100 U/ml of rhIL-2, and plated in 96-well flat-bottom plates
(Falcon Labware, Lincoln Park, NJ) at 105 cells/0.2 ml
using six replicates/group. Cultures were passed every 3–4 days by
removing one-half the volume of medium and replacing it with fresh
complete medium containing 100 U/ml of rhIL-2. The culture fluids that
were removed were then monitored for particle-associated reverse
transcriptase activity (24). In some experiments, the anti-CD3 and
CD28 Ab beads were added to the cells every 7–10 days at a
bead-to-cell ratio of 2:1.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Initially, we determined the effect that triggering the CD28
molecule on CD4+ cells during CD3 stimulation would have on
the sensitivity of these cells to infection with HIV. Purified
CD4+ cells from HIV-seronegative donors were treated with
magnetic beads coated with both anti-CD3 and CD28 Ab. As controls,
CD4+ cells from the same donors were exposed to beads to
which only anti-CD3 Ab or anti-CD28 Ab was coupled. Following
stimulation, the CD4+ cells were infected with 100
TCID50 of either the M-tropic HIV-1SF162 or the
TCL-tropic HIV-1SF33 isolate. The results indicated both
types of HIV strains could replicate in the CD28-costimulated
CD4+ cells (Fig. 2). Peak HIV
replication of both strains (645 x 103 and 840
x 103cpm/ml, respectively) was observed 10 days following
the initiation of infection. Exposure to anti-CD3 Ab coupled to
beads alone led to moderate (146 x 103 cpm/ml) levels
of HIV-1SF162 replication 10 days after infection (Fig. 2A). Moreover, at the time of infection ß-chemokine
production was elevated in CD28-costimulated cells (112 ng/ml MIP-1,
63 ng/ml MIP-1ß, and 7 ng/ml RANTES) compared with the same cells
stimulated with anti-CD3 Ab beads alone (67.9 ng/ml MIP-1, 37.8
ng/ml MIP-1ß, and 3.2 ng/ml RANTES) Ab.
|
B). Both strains of HIV-1 did
not replicate to detectable levels in CD4+ cells exposed to
anti-CD28 Ab beads alone (Fig. 2). HIV replication in CD4+ cells costimulated with the CD80 and CD86 molecules
Because engagement of the CD28 molecule with anti-CD28
Ab during stimulation enhanced the replication of HIV in
CD4+ cells, we next determined if the natural ligands
(i.e., the CD80 and CD86 molecules) for the CD28 receptor could replace
these Abs in the costimulation process. CD4+ cells from
HIV-seronegative individuals were cocultured with beads coated with
both anti-CD3 Ab plus CD80Ig and anti-CD3 Ab plus CD86Ig before
infection with HIV-1SF162. As a control, beads coated with
anti-CD3 and anti-CD28 Ab together were used to stimulate the
CD4+ cells. Exposure of CD4+ cells to CD80 and
CD86 molecules during stimulation with anti-CD3 Ab led to
substantial production of HIV-1SF162 (Fig. 3). The levels of virus replication were
similar to the levels observed from CD4+ cells stimulated
with anti-CD3 and CD28 Ab beads.
|
Treatment of CD4+ cells from HIV-infected individuals
with immobilized anti-CD3 and CD28 Abs ex vivo has been reported to
decrease endogenous virus production and proviral DNA to
undetectable levels (10). Therefore, we determined if triggering the
CD28 molecule during stimulation would affect the production of virus
from naturally infected CD4+ cells from HIV-seropositive
individuals. One of the subjects studied was clinically healthy and
infected with a dominant NSI virus (Fig. 4A); the other individual had
an AIDS diagnosis and was infected with an SI virus strain (Fig. 4B) (see Materials and Methods). CD4+
cells from both HIV-infected individuals were exposed to beads to which
either anti-CD3 Ab alone or anti-CD3 Ab together with
anti-CD28 Ab were coupled. CD28 costimulation of CD4+
cells from both HIV-infected individuals increased virus production
over that seen with CD4 cells stimulated with anti-CD3 Abs alone
(Fig. 4). As expected, the CD4+ cells from the AIDS patient
(Fig. 4B) released virus sooner than the cells from the
asymptomatic individual (Fig. 4A).
|
Our current findings that CD28-costimulation induces higher levels
of HIV replication differs from those of a recent study by other
investigators that reported CD28-costimulation blocks HIV replication
(10, 11). In examining the possible reason(s) for the difference, we
evaluated whether constant exposure of CD4+ cells to the Ab
beads would change the dynamics of virus infection. This procedure was
used because other investigators, when studying the effects of
CD28-costimulation on HIV production by infected CD4+
cells, added the anti-CD3 and CD28 Ab beads to their culture
whenever the CD4+ cell size decreased (10, 14).
CD4+ cells were isolated from HIV-seronegative individuals
and stimulated for 3 days with anti-CD3 and CD28 Ab coupled to
beads. The cell-bead mixture was then split into two groups; the beads
were either removed or left in culture during acute infection. HIV
could be detected within 7–10 days in culture when the beads were
removed from the cells before virus inoculation (Fig. 5). HIV replication was delayed for up to
7 days when the beads were present with the cells during virus
infection (data not shown). Furthermore, the addition of fresh
anti-CD3 and CD28 Ab beads every 7–10 days following infection led
to prolonged suppression of HIV replication (Fig. 5), even as long as 5
wk following infection. Only after removal of the beads (e.g., 25 days
postinfection) did virus production take place (Fig. 5B).
The kinetics of virus production from these cells was similar to that
observed with the infected cells in which the beads were initially
removed. These findings indicate that continual CD28-costimulation can
suppress the production of HIV by infected CD4+ cells.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). This result
was observed whether anti-CD28 Ab or the natural ligands for CD28
(CD80 or CD86) (Fig. 3) were used to trigger the CD28 molecule.
Moreover, the enhanced HIV replication (both NSI and SI
phenotype) was noted following similar CD28 costimulation of
naturally infected CD4+ cells from HIV-seropositive
individuals (Fig. 4). This finding is in agreement with the studies by
Smithgall et al. (9), who demonstrated that optimal production of HIV
from CD8+ cell-depleted PBMC of asymptomatic HIV-infected
individuals occurred when accessory cells bearing CD80 and CD86 were
present during stimulation.
Our findings differ from those of Levine et al. (10), which showed that
activation of CD4+ T lymphocytes with immobilized
anti-CD3 and CD28 Ab induced resistance to productive infection by
M-tropic viruses. As stated above, using anti-CD3 and CD28 Ab
coupled to beads (including those obtained from these authors; data not
shown), we found that the CD28-costimulated CD4+ cells were
susceptible to acute infection by M-tropic viruses. The effect of
CD28-costimulation of CD4+ cells on acute infection by the
TCL-tropic strain appeared to be minimal, which probably reflects the
rapid growth kinetics of this strain of HIV (21). Importantly, we noted
that the differences observed between the two studies were based on
whether or not the anti-CD3 and CD28 Ab beads were removed from the
CD4+ cells after stimulation. In the experiments conducted
by Levine et al., the anti-CD3 and CD28 Ab beads were left with the
cells during the entire culture period. Furthermore, Ab beads were
added to the CD4+ cells when their cell size decreased to
those of resting cells (10, 14). In our studies, removing the beads
immediately after stimulation led to virus production within 7–10 days
postinfection, whereas no HIV replication was observed when the beads
were left on and replenished (Fig. 5). The beads had to be added to the
cells every 7–10 days or else the virus was released in culture (data
not shown). Even when the beads were removed from the cells 25 days
after prolonged CD28-costimulation, virus was still recoverable (Fig. 5B). The virus replication, two passages after removal of
the beads, was similar to that observed after the initial infection of
cells in which beads were removed after stimulation, before acute
infection (Fig. 5B). These findings suggest that HIV can
infect CD4+ cells constantly exposed to anti-CD3 and
CD28 Ab beads but virus production occurs only after the source of CD28
costimulation is removed.
Other investigators have demonstrated that CD28 costimulation of CD4+ cells enhances ß-chemokine production and loss of CCR5 expression (11, 12). Both processes can prevent entry of M-tropic viruses into the cell and virus production (13). Our studies suggest that CD28 costimulation does not effectively block the initial infection of CD4 cells by the M-tropic virus; however, further spread of the virus in the cultures appears to be prevented. We have observed, in preliminary studies, that the CD28 costimulation leads to an increase in ß-chemokine levels in the culture fluid. Only by maintaining the CD28 costimulation of CD4+ cells through the addition of Ab beads every 7–10 days are high levels of ß-chemokines maintained (our unpublished observations). Thus, our results support those of others (11) suggesting a relationship between high ß-chemokine production and reduced virus replication in the cultured CD4+ cells. Further studies are underway to evaluate the effect of constant CD28 costimulation on infection of CD4+ cells by both NSI and SI strains. Whether therapeutic approaches can be developed to mimic these conditions of costimulation in vivo remains to be determined.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Department of Microbiology and Immunology, State University of New York Health Science Center, Syracuse, NY 13210.
3 Address correspondence and reprint requests to Dr. Jay A. Levy, Department of Medicine, University of California, San Francisco, CA 94143-1270.
4 Abbreviations used in this paper: M-tropic, HIV strain able to infect macrophages; TCL-tropic, HIV strain able to infect T cell lines; rh, recombinant human; TCID50, 50% tissue culture infectious dose; CMF-HBSS, calcium- and magnesium-free HBSS; SI, syncytium-inducing; NSI, nonsyncytium-inducing.
Received for publication May 13, 1998. Accepted for publication August 7, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  F. Gondois-Rey, A. Biancotto, M. A. Fernandez, L. Bettendroffer, J. Blazkova, K. Trejbalova, M. Pion, and I. Hirsch R5 Variants of Human Immunodeficiency Virus Type 1 Preferentially Infect CD62L- CD4+ T Cells and Are Potentially Resistant to Nucleoside Reverse Transcriptase Inhibitors J. Virol., January 15, 2006; 80(2): 854 - 865. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Schweighardt, A.-M. Roy, D. A. Meiklejohn, E. J. Grace II, W. J. Moretto, J. J. Heymann, and D. F. Nixon R5 Human Immunodeficiency Virus Type 1 (HIV-1) Replicates More Efficiently in Primary CD4+ T-Cell Cultures Than X4 HIV-1 J. Virol., September 1, 2004; 78(17): 9164 - 9173. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Cook, L. Albacker, A. August, and A. J. Henderson CD28-dependent HIV-1 Transcription Is Associated with Vav, Rac, and NF-{kappa}B Activation J. Biol. Chem., September 12, 2003; 278(37): 35812 - 35818. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Beck, M. B. Blackmon, C. M. Rose, S. L. Kimzey, A. M. Preston, and J. M. Green T Cell Costimulatory Molecule Function Determines Susceptibility to Infection with Pneumocystis carinii in Mice J. Immunol., August 15, 2003; 171(4): 1969 - 1977. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Cook, A. August, and A. J. Henderson Recruitment of Phosphatidylinositol 3-Kinase to CD28 Inhibits HIV Transcription by a Tat-Dependent Mechanism J. Immunol., July 1, 2002; 169(1): 254 - 260. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Mengozzi, M. Malipatlolla, S. C. De Rosa, L. A. Herzenberg, L. A. Herzenberg, and M. Roederer Naive CD4 T cells inhibit CD28-costimulated R5 HIV replication in memory CD4 T cells PNAS, September 13, 2001; (2001) 211205098. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Creson, A. A. Lin, Q. Li, D. F. Broad, M. R. Roberts, and S. J. Anderson The Mode and Duration of Anti-CD28 Costimulation Determine Resistance to Infection by Macrophage-Tropic Strains of Human Immunodeficiency Virus Type 1 In Vitro J. Virol., November 1, 1999; 73(11): 9337 - 9347. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Mengozzi, M. Malipatlolla, S. C. De Rosa, L. A. Herzenberg, L. A. Herzenberg, and M. Roederer Naive CD4 T cells inhibit CD28-costimulated R5 HIV replication in memory CD4 T cells PNAS, September 25, 2001; 98(20): 11644 - 11649. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
; A and B) or
anti-CD3 and CD28 Abs (
; B) were bound. Tritiated
thymidine was added the last 18 h of incubation. The data
represent the mean incorporation of tritiated thymidine by
CD4+ cells cultured in triplicate with various numbers of
beads. The results are representative of two studies.
), anti-CD28 (
), or both Abs (•) were bound.
The cells were then infected with 100 TCID50 of
HIV-1SF162 (A) or HIV-1SF33 (B) and cultured
(105 cells/well) for 13 days. Every 3–4 days culture
fluids were removed and monitored for reverse transcriptase activity
(24). The data are the mean of results from six replicate cultures. The
results are representative of two separate experiments using different
donors.
