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* Center for Neurovirology and Neurodegenerative Disorders, and Departments of Pathology and Microbiology and
Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198; and
Program in Molecular Immunology, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ELISPOT.
CD8, granzyme B, HLA-DR, and CD45R0 Ag-reactive T cells and
CD79
-positive B cells migrated to and were in contact with human MDM
in brain areas where infected macrophages were abundant. The numbers of
productively infected MDM were markedly reduced (>85%) during 2 wk of
observation. The human PBL-NOD-SCID HIVE mouse provides a new tool for
studies of cellular immune responses against HIV-1-infected brain
mononuclear phagocytes during natural disease and after
vaccination. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, -
, and - and
cytokines (TGF- and TNF-) among others (7, 8). In
addition, Ag-specific CD8+ CTL serves a prominent
role in the control of HIV infection, in both brain and peripheral
tissues (9, 10). HIV-1-infected tissue macrophages attract
T and B lymphocytes while serving as APC, thus affecting virus-specific
acquired immune responses. In this way, HIV-1-specific CTL can
eliminate virus in tissues such as brain, where ongoing viral
production occurs.
To test the ability of HIV-1-infected MP to induce an effective CTL
response as a means of viral elimination, we produced a mouse model of
HIV-1 encephalitis (HIVE) in nonobese diabetic (NOD)-C.B-17 SCID mice
reconstituted with human (hu) PBL (hu-PBL-NOD-SCID HIVE). HIVE was
established 7 days after immune reconstitution by stereotactic
injection of human HIV-1-infected human monocyte-derived macrophages
(MDM) into the subcortex. Tetramer staining showed HIV-1gag- and
HIV-1pol-specific CTL in mouse spleens 1 wk after injection of infected
MDM. CD8, granzyme B, HLA-DR, and CD45R0 Ag-immunoreactive T cells and
CD79-positive B cells migrated to the sites of human MDM. This
hu-PBL-NOD-SCID HIVE mouse model recapitulates the cellular immune
responses against HIV-1-infected brain macrophages that occur in humans
during progressive disease. The generation of HIV-1-specific CTL
against virus-infected human macrophages in mice was demonstrated in
this report. The work supports the hypothesis that elimination of
infected HIV-1 macrophages can occur effectively in the nervous system
and provides new insights into the mechanisms of restricted virus
infection in the brain that occurs over years following viral
exposure.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monocytes and lymphocytes were obtained from leukopheresis of HIV-1-, HIV-2-, and hepatitis B-seronegative donors and were purified by countercurrent centrifugal elutriation. Cell suspensions were documented as >98% monocytes by criteria of cell morphology in Wright-stained cytosmears. Monocytes were cultured as suspensions in Teflon flasks (2 x 106 cells/ml) in DMEM (Sigma-Aldrich, St. Louis, MO) with 10% heat-inactivated pooled human serum, 1% glutamine, 50 µg/ml gentamicin, and/or 10 µg/ml ciprofloxacin (Sigma-Aldrich) and 1000 U/ml highly purified human rM-CSF (a gift from Genetics Institute, Cambridge, MA). Culture medium was changed every 3 days. All tissue culture reagents were screened and found negative for endotoxin (<10 pg/ml; Associates of Cape Cod, Woods Hole, MA) and mycoplasma contamination (Gen-Probe II; Gen-Probe, San Diego, CA).
HIV-1 infection of MDM
After 7 days in culture, MDM were infected with HIV-1ADA (a macrophage tropic strain) at a multiplicity of infection of 0.01 (11). The percentage of HIV-infected MDM was determined by immunostaining with HIV-1 p24gag mAb (DAKO, Carpinteria, CA).
NOD-SCID mouse model of HIVE
Four-week-old male NOD-C.B-17 SCID mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were maintained in sterile microisolator cages under pathogen-free conditions in accordance with ethical guidelines for care of laboratory animals at the University of Nebraska Medical Center set forth by the National Institutes of Health. All animal manipulations, including intracerebral (i.c.) inoculations, were performed in a laminar flow hood. Animals were injected i.p. with asialo-GM1 polyclonal rabbit Abs (WAKO, Richmond, VA) 24 h before and 3 days after PBL injection to facilitate engraftment.
Human PBMC from HLA-A*0201-positive donor were separated into a monocyte- and PBL-enriched fraction. Human PBL (8 x 107 cells in 0.5 ml PBS) were injected i.p. into recipient animals. Three of 16 injected mice did not engraft human lymphocytes. Injection of HIV-1ADA-infected MDM into the subcortex was performed following i.p. ketamine/xylazine anesthesia (100 mg/kg ketamine and 16 mg/kg xylazine) on day 8 after PBL engraftment. These were named hu-PBL-NOD-SCID HIVE mice. Control NOD-SCID mice not engrafted with human PBL, but that received i.c. injections of HIV-1ADA-infected MDM, were termed NOD-SCID HIVE mice. Each animal received 10 µl of suspension containing 3.0 x 105 HIV-1-infected MDM injected into the left hemisphere caudate and putamen, using the coordinates previously described (12). Animals were sacrificed 7, 14, and 21 days after injection. To evaluate the optimal conditions for generating a CTL response, 11 mice/group received simultaneous transplantation of HLA-mismatched PBL (i.p.) and HIV-1-infected MDM (i.c.). These animals were sacrificed at 3, 7, and 14 days after cell injections. Blood was collected in EDTA-containing tubes, and plasma was separated for HIV-1p24gag ELISA tests (Beckman Coulter, Miami, FL).
Activation and expansion of human cells from the spleens of NOD-SCID mice
Single-cell suspensions were prepared from spleens depleted of erythrocytes. Splenocytes were cultivated at a concentration of 2 x 106 cells/ml in RPMI 1640 (Sigma-Aldrich) supplemented with 10% heat-inactivated FBS (Mediatech; Cellgro, Herndon, VA), 1% L-glutamine, and 0.2% gentamicin. Single-cell suspensions were activated with PHA-P (1 µg/ml) in RPMI 1640 supplemented with 10% IL-2 (Advanced Biotechnologies, Columbia, MD). On day 11 after stimulation, cells were stained and free of mouse CD45+ (leukocyte common Ag, Ly-5) contamination.
Immunophenotypic analyses
Splenocytes from mice engrafted with human PBL were incubated with fluorochrome-conjugated mAbs to human CD4, CD8, and CD56 (BD PharMingen, Los Angeles, CA) for 30 min at 4°C. FITC-conjugated mAbs to mouse CD45 (leukocyte common Ag, Ly-5) identified murine cells. To determine Ag-specific CTL, allophycocyanin-conjugated tetramer staining for HIV-1gag (p17(aa 77–85) SLYNTVATL, SL-9) and HIV-1pol ((aa 476–485) ILKEPVHGV, IL-9) was performed on fresh and PHA/IL-2-stimulated splenocytes. Cells were stained following the recommendations of the National Institutes of Health/National Institute of Allergy and Infectious Disease, National Tetramer Core Facility (Atlanta, GA), and analyzed with a FACSCaliber using CellQuest software (BD Immunocytometry Systems, San Jose, CA).
Tetramer staining was positive in 0.0–0.04% of nonactivated splenocytes derived from hu-PBL-NOD-SCID mice not exposed to HIV-1. Staining of PHA/IL-2-activated lymphocytes derived from PBL-reconstituted animals not exposed to HIV-1 was 0.22–0.49% for HLA-A*0201 and 0.07–0.14% for HLA-mismatched donor PBL. Splenocytes from animals engrafted with HLA-A3-mismatched donor PBL and PHA/IL-2-stimulated lymphoblasts served as controls (data not shown).
ELISPOT assay
Human IFN- ELISPOT assays were performed from single-cell
suspensions of splenocytes collected from hu-PBL-NOD-SCID HIVE mice.
Human PBL not exposed to HIV-1 served as the control. Lymphocytes were
isolated from splenocyte single-cell suspensions by gradient
centrifugation and adjusted to a concentration 2 x
106 cells/ml. Trypan blue exclusion showed 98%
cell viability. ELISPOT was performed in triplicate determinations (50
µl of responders) in nitrocellulose-lined 96-well microtiter plates
(MAHA S45; Millipore, Bedford, MA) with a human IFN- ELISPOT kit
(Cell Science, Norwood, MA). The assays were performed according to
manufacturer’s instructions. Irradiated syngeneic donor PBL (50 µl,
with a ratio to responders of 1:1) served as APCs. Ags, including
HIV-1gag (SL-9), HIV-1pol (IL-9), and OVA, were used at a concentration
of 10 µg/ml. Activation with PHA at a concentration 1 µg/ml was
used as a positive control. Results were adjusted to the number of
human cells in the suspension determined by FACS.
Histopathology and image analysis
Brain tissue was collected at necropsy. Tissue was fixed in 4%
phosphate-buffered paraformaldehyde and paraffin-embedded or frozen at
-80°C for later analysis. Neuropathological analyses were performed
7, 14, and 21 days after injection of infected MDM. For each mouse
30–100 serial (5-µm-thick) sections were cut from the injection site
and three to seven sections (10 sections apart) around the needle track
were analyzed. Blocks were cut until the inoculation site was
identified. The Abs used for immunohistochemical tests are listed in
Table I
. The Vectastain Elite ABC kit
(Vector Laboratories, Burlingame, CA) and the Dako EnVision kit
were the detection system for one or two Ags. All sections were
counterstained with Mayer’s hematoxylin. Deletion of the primary Ab or
use of mouse IgG served as the control. The numbers of HIV-1
p24gag Ag-positive MDM and lymphocytes detected
were averaged for each mouse. Stained tissue images were analyzed with
a Nikon Microphot-FXA microscope (Melville, NY) and acquired by
DVC-1312 digital optics. Data was analyzed with Excel (Microsoft,
Redmond, WA) and Kaleidagraph 2.0 software (Synergy Software, Reading,
PA) using the Student t test for comparisons.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice engrafted with PBL from HLA-A*0201-positive donors injected
i.p. generated hu-PBL-NOD-SCID animals. Seven days after lymphocyte
engraftment HIVE was established by stereotactic injection of syngeneic
human HIV-1ADA-infected MDM into the basal
ganglia and caudate, generating hu-PBL-NOD-SCID HIVE mice.
Neuropathological analyses were performed 7, 14, and 21 days after
injection of infected MDM (Fig. 1).
Immunostaining of brain sections for glial fibrillary acidic
protein (GFAP) and Griffonia simplicifolia showed
wide areas of reactive astrocytes and microglia in and around areas of
infected human cells (MDM and lymphocytes) 7 days after injection of
human MDM. In hu-PBL-NOD-SCID HIVE mice, MDM showed elongated processes
with dendritic-like morphology in contact with lymphocytes. Such
morphological changes in MDM were rare at 14 and 21 days. MDM uniformly
immunoreactive for HLA-DR and surrounded by HLA-DR Ag-positive
lymphocytes were readily observed within the perivascular spaces.
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Lymphocyte migration into brain was significant 7 days after
injection of MDM (Fig. 2). On the
average, 303.8 ± 90.0 lymphocytes were detected in each 5-µm
section (Table II). Most lymphocytes were
in close proximity to human MDM, in or around the needle track, and in
the surrounding brain parenchyma adjacent to microvessels. Few
lymphocytes were detected in vascular lumens in the contralateral
noninjected hemisphere. Fifty to 80% of lymphocytes were
CD8+, and 15–45% were granzyme B-positive on
day 7 after MDM injection. On day 14, the number of lymphocytes in the
brain decreased 2-fold to 151.6 ± 50.1 cells/section. The ratio
of MDM to lymphocytes was 1:20, the maximum throughout the experimental
period. On day 21, 35.3 ± 4.2 lymphocytes/section were observed.
At all time points, lymphocytes were present only in areas with
infected MDM (including the meninges, choroid plexus, and ventricles).
The majority of lymphocytes were CD45RO-immunoreactive, indicating that
they were activated memory cells. CD79-positive B cells were also
found in the injected hemisphere and in the meninges, but did not
exceed 1–5% of the migrated lymphocytes. HIV-1
p24gag-immunoreactive lymphocytes were readily
detected in brain 7 days after MDM injection. HIV-1 p24
gag-positive syncytia was observed at the site of
MDM injection as well as in perivascular spaces and meninges. The
numbers of infected lymphocytes were 39.0 ± 9.8, 14.5 ±
7.5, and 0.6 ± 0.4% on days 7, 14, and 21 following MDM
injection, respectively.
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One week after injection, the average numbers of human MDM were
29.1 ± 11.6 and 47.2 ± 14.8/section in NOD-SCID HIVE and
hu-PBL-NOD-SCID HIVE mice, respectively. The percentages of HIV-1
p24gag Ag-reactive MDM were 74.7 ± 1.5 and
90.6 ± 5.4% in the NOD-SCID HIVE and hu-PBL-NOD-SCID HIVE mice,
respectively. On day 14, the numbers of macrophages per section were
51.0 ± 11.8 and 6.6 ± 2.3 in the NOD-SCID HIVE and
hu-PBL-NOD-SCID HIVE mice, respectively (p <
0.01; Fig. 3). All MDM in the
hu-PBL-NOD-SCID HIVE and 65.9 ± 17.7% of the NOD-SCID HIVE MDM
expressed HIV-1 p24gag Ag. On day 21, three of
four hu-PBL-NOD-SCID HIVE mice had no or few multinucleated giant cells
(0.4 ± 0.2/section). In NOD-SCID HIVE mice, the number of MDM was
15.2 ± 5.5/section. Of these, 83.3 ± 7.1% were viral
Ag-positive (Table II
). The apoptotic macrophages and lymphocytes were
visible in affected brain tissue of hu-PBL-NOD-SCID HIVE mice on day 7
after MDM injection. Multinucleated HIV-1-p24 giant cells were also
seen without evidence of apoptosis in both animal groups (data not
shown).
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Tetramer staining was used to determine whether the engrafted
human lymphocytes from HIV-1 and HIV-2-seronegative HLA-A*0201-positive
donors reacted to HIV-1-infected brain macrophages. In these assays,
freshly prepared and PHA/IL-2-activated splenocytes (both containing
human lymphocytes) were used. Two well-characterized
HLA-A*0201-restricted immunodominant CTL epitopes (HIV-1gag (SLYNTVATL)
and HIV-1pol (ILKEPVHGV)), found in a majority of HIV-1-seropositive
patients, were tested. Three-color flow cytometric analysis using
tetramer-allophycocyanin, anti-human CD8-PE, and
anti-mouse-Ly-5-FITC were performed. CD8+/HIV-1gag- and
CD8+/HIV-1pol-positive human cells were
detectable, but rare, in splenocyte suspensions. The percentages of
human HIV-1 specific CD8+ cells following
PHA/IL-2 activation of mouse splenocytes were 1.4 ± 0.3, 4.8
± 0.7, and 3.7 ± 1.5% (days 7, 14, and 21) and 0.7 ± 0.1,
1.8 ± 0.3, and 4.0 ± 1.9% (days 7, 14, and
21) for HIV-1gag and HIV-1pol,
respectively (Table III and Fig. 4). The
numbers of MDM present in brain inversely correlated with the numbers
of CD8+ HIV-1gag cells in spleen on day 7, but
not on days 14 and 21 (Tables II and III).
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ELISPOT
To confirm functional properties of HIV-1-specific human cells
that repopulated spleens of hu-PBL-NOD-SCID HIVE mice, an IFN-
ELISPOT was performed with collected frozen samples. Flow cytometry
demonstrated that 30% of the cells were human T lymphocytes, and 80%
of them were CD8-positive cells. Cells were activated with PHA (1
µg/ml), SL-9, IL-9 peptides, and OVA in the presence of irradiated
syngeneic PBL. Results are shown in Fig. 5. Activation with PHA induced >1500
spots. The number of IFN--producing cells increased after exposure
to HIV-1gag (SL-9) compared with those cells not exposed to the HIV-1
peptide (p < 0.05).
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Viral production was monitored by determining levels of
HIV-1p24gag Ags in plasma of the NOD-SCID HIVE
and hu-PBL-NOD-SCID HIVE mice on days 14 and 21. HIV-1
p24gag was not found in NOD-SCID HIVE mice.
However, mice engrafted with lymphocytes had plasma HIV-1
p24gag Ag levels of 1043 ± 509 and 598
± 187 pg/ml on days 14 and 21 (Table III).
Numbers of human CD4-, CD8-, and CD56-immunopositive cells in
splenocytes of hu-PBL-NOD-SCID HIVE mice were next analyzed. A marked
decrease in the number of human CD4+ T
lymphocytes was observed on day 21 after HIV-1-infected MDM injection.
The percentages of CD4+ T cells in spleen were
8.8 ± 1.7, 8.7 ± 1.7, and 0.7 ± 0.1% on days 7, 14,
and 21, respectively. In contrast, the percentages of
CD8+ T cells were 20.3 ± 4.3, 28.4 ±
7.7, and 23.2 ± 5.4% on days 7, 14, and 21, respectively. The
proportion of NK cells (CD56+) was highest on day
7 (13.3 ± 2.7%), then decreased on days 14 and 21 (2.4 ±
0.8 and 2.0 ± 0.4%, respectively). On day 7 after MDM injection,
the number of NK and CD8+ cells in spleen
inversely corresponded to the number of human brain MDM (Table II, III).
HIV-1-infected MDM were not detected in peripheral tissue. However,
beginning on day 14 and continuing until day 21, HIV-1
p24gag-immunopositive lymphocytes and lymphocyte
syncytia surrounded by CD8+ T cells were detected
in liver, lung, and lymph nodes. These cells had typical lymphocyte
morphology and failed to express CD68 Ags (data not shown). This
corresponded to increased levels of HIV-1 p24gag
in peripheral blood.
HLA-mismatched cells
As an additional control for the generation of HIV-1-reactive
cells, cellular immune responses against the human MDM were monitored
after simultaneous transplantation of HLA-mismatched PBL (i.p.) and MDM
(i.c.). Neither migration of lymphocytes into brain nor reduction of
MDM was observed (Table IV). Only a
transient decrease in the numbers of HIV-1-infected MDM in
hu-PBL-NOD-SCID HIVE, compared with those in NOD-SCID HIVE animals
(0.7 ± 0.5 and 34.9 ± 9.9%, respectively;
p < 0.005), was observed on day 7. There was no
statistically significant difference in the numbers of MDM in either
group of animals (34.8 ± 16.6 and 38.8 ± 15.6 MDM/section,
respectively) on day 14. The numbers of CD4+ and
CD8+ cells in spleens were equivalent in animals
on days 7 and 14 after PBL transplantation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The assault of HIV-1 on the nervous system is one of the most significant clinical consequences of progressive viral infection in its human host. Certainly, immune control of viral infection is involved in the delay of onset of productive HIV-1 replication in brain. How this occurs and why only a portion of those infected succumb to HIV-1-associated dementia remain incompletely understood. In this report, we demonstrate that viral infection of brain macrophages is itself a strong inducer of CTL and a powerful attractant for lymphocyte infiltration into the brain. The enhanced cell trafficking of HIV-1 Ag-specific CD8+ T lymphocytes into brain leads to the rapid elimination of virus-infected macrophages. We propose that similar mechanisms are operative during the course of viral infection in humans. This serves to protect the brain throughout most of HIV-1 disease or until significant immunosuppression leads to unrestricted viral growth and encephalopathy.
This work is among the first to generate a de novo human CTL response against HIV-1-infected cells in mice. In this system, HIV-1-infected MDM served as a stable reservoir for virus and a powerful APC in brain for the generation of HIV-1-specific CTL responses. Previous studies showed that engraftment of human PBL into NOD-SCID mice for 1–4 wk did not result in T cell anergy (15). Human T lymphocytes have been shown to affect humoral and cellular anti-viral immune responses in SCID and NOD-SCID mice following injection of HIV-1 proteins (16) or responses by dendritic cells pulsed with inactivated virus (17). However, HIV-1-specific CTL have not been generated in rodents by productively infected human cells. This observation makes the hu-PBL-NOD-SCID HIVE mouse model unique for studies of acquired viral immunity and for testing vaccine candidates.
CTL responses against HIV-1-infected human MDM were shown by tetramer staining of HIV-1gag- and HIV-1pol-specific CD8+ in spleens of hu-PBL-NOD-SCID HIVE mice. Virus-specific CD8+ cells were not found in animals injected i.c. with uninfected MDM, nor were these cells eliminated. Importantly, the number of tetramer-positive cells in spleen increased up to day 14 after injection of HIV-1-infected MDM. Thereafter, the number of HIV-1gag-specific CTL was minimally decreased in spleen with the depletion of human CD4+ T lymphocytes. In addition, the appearance of HIV-1-specific CTL in spleen corresponded to the presence of CD8+/granzyme B-positive lymphocytes in brain 7 days after injection of HIV-1-infected MDM. Infiltrating CD8+, CD45RO+ lymphocytes were observed in contact with MDM and were associated with a reduction in the number of infected cells. Indeed, the number of tetramer-positive cells in spleen was highest 14 days after human MDM injection, while the number of MDM in brain was markedly decreased (1 macrophage:20 lymphocytes at this time point). Lastly, we demonstrated a progressive loss of human CD4+ T lymphocytes in the spleens of hu-PBL-NOD-SCID HIVE mice. However, complete abrogation of CD4+ T lymphocytes was not observed. Indeed, the CTL responses seen would be impossible without the presence of residual helper T lymphocytes responding to MP-presented Ags.
Interestingly, our data showed that human MDM remain potent APC even
when placed within the murine brain environment.
HIV-1ADA infection of brain MDM and the presence
of syngeneic lymphocytes induced morphologic changes reminiscent of
what occurs after IFN- treatment and/or during Ag presentation
(18). One-third of freshly elutriated human monocytes are
CD2+ precursors of myeloid lineage dendritic
cells (19). We cannot exclude the direct involvement of
dendritic cells engrafted with lymphocytes in the development of immune
responses. However, after 1 wk in a xenogenic environment, few immature
precursors of dendritic cells would survive. It is more likely that
human MDM would adopt a dendritic morphology with potent APC functions.
In this scenario, in vitro maturation of MDM in the presence of MCSF
was not a limiting factor for generating HIV-1-specific CTL responses
(20, 21). MDM trafficking between the brain and periphery
also could trigger HIV-1-specific CTL responses (22). Our
observation supports a previously published report for SIV encephalitis
(23). In this report, SIV-specific
CD8+ CTL were isolated from blood, cerebrospinal
fluid, and brains of SIV-infected rhesus macaques. CTL were found as
early as 1 wk following viral exposure and correlated with SIV p27 Ags
in blood. CTL isolated from cerebrospinal fluid and/or brain recognized
a unique set of viral proteins compared with CTL from blood. This
suggested that either a unique migratory pattern to the CNS occurred or
a difference in activation profiles of lymphocytes emerged in these
tissue compartments.
Our model system recapitulates what occurs in human HIVE, where productively infected brain macrophages recruit lymphocytes into the CNS. Such leukocyte trafficking could serve both as a means of viral spread as well as an attempt to elicit cellular immune responses to destroy infected MP. Certainly, several pathways are operative by which lymphocytes may reach the brain (24, 25). Circulating lymphocytes from periphery to brain and vice versa may deliver virus to regional lymph nodes as well as distant lymphoid tissues where uninfected T lymphocytes circulate (26). Ultimately, the forces that restrain viral growth prevail, resulting in restricted virus production. However, this does not occur without some cost to the host. Indeed, in our model, HIV-1 spreads from brain to periphery despite rapid elimination of infected cells. Viral p24gag protein levels were easily detected in the plasma of hu-PBL-NOD-SCID HIVE animals and corresponded to the rapid depletion of human CD4+ T lymphocytes from spleen within 3 wk after i.c. injection of infected macrophages. The importance of bidirectional migration of macrophages and lymphocytes in the induction of acquired immune responses within the brain is under investigation.
Cell-to-cell interactions between brain macrophages and lymphocytes represent an ongoing process leading to the elimination of infected cells. This process includes cell trafficking, Ag presentation, and cell destruction. The relative contributions of these individual events to the overall process and the roles of NK, NK-T, and CD4+ T lymphocytes in virus-infected macrophage elimination requires further study.
There are several parts of this model system worth noting: 1) the generation of CTL responses, which occurred when NOD-SCID animals were reconstituted with human PBL then injected i.c. with virus-infected macrophages 7 days later; 2) the requirement for M-CSF to human MDM, which permitted both optimal cell differentiation and viral growth; and 3) the timing of the lymphocyte reconstitution and their properties. Indeed, the simultaneous placement of PHA/IL-2-activated lymphoblasts i.p. and HIV-1-infected MDM i.c. produced low levels of infiltrating T cells. Moreover, the simultaneous placement of HLA-mismatched PBL and MDM failed to generate an effective anti-retroviral immune response. CTL escape mutants are one possible concern for long-term utility of this acute system in its relevance to human disease (27). Other concerns center around the reconstitution system and the injection of infected cells and their ability to elicit disease. Despite the limitations inherent in this animal model system, the ability to generate in vivo CTL responses against infected cells provides an avenue for studies of the effector arm of immune responses, vaccine development, and the discovery of drugs that target productively infected brain MP.
Four features of natural HIV-1 infection were reproduced in this model system, including 1) production of virus in both brain and blood, 2) depletion of human CD4+ T lymphocytes, 3) generation of HIV-1-specific CD8+ CTL, and 4) restriction of ongoing viral brain infection by CTL-mediated removal of infected cells. The ability to measure human primary immune responses against human infected brain macrophages in a murine system of HIVE is unique. Indeed, this hu-PBL-NOD-SCID HIVE model should prove valuable in elucidating how T cell immune responses regulate viral growth in brain macrophages and aid in the development and testing of an effective vaccine against HIV-1.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Larisa Y. Poluektova, Center for Neurovirology and Neurodegenerative Disorders, 985215 Nebraska Medical Center, Omaha, NE 68198-5215.
3 Y.P. and H.E.G. contributed equally to this work.
4 Abbreviations used in this paper: MP, mononuclear phagocyte; HIVE, HIV-1 encephalitis; i.c., intracerebral; MDM, monocyte-derived macrophage; NOD, nonobese diabetic; hu, human; GFAP, glial fibrillary acidic protein; DAB, diaminobenzidene.
Received for publication September 24, 2001. Accepted for publication January 25, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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interferon, tumor necrosis factor (TNF-), and TNF- when they encounter their target antigens. J. Virol. 67:2844. suppresses human immunodeficiency virus expression and replication in infected cells of the monocyte/macrophage lineage. J. Exp. Med. 173:589.-chain. Clin. Exp. Immunol. 122:117.[Medline]
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R. Potula, L. Poluektova, B. Knipe, J. Chrastil, D. Heilman, H. Dou, O. Takikawa, D. H. Munn, H. E. Gendelman, and Y. Persidsky Inhibition of indoleamine 2,3-dioxygenase (IDO) enhances elimination of virus-infected macrophages in an animal model of HIV-1 encephalitis Blood, October 1, 2005; 106(7): 2382 - 2390. [Abstract] [Full Text] [PDF]
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S. Gorantla, K. Santos, V. Meyer, S. Dewhurst, W. J. Bowers, H. J. Federoff, H. E. Gendelman, and L. Poluektova Human Dendritic Cells Transduced with Herpes Simplex Virus Amplicons Encoding Human Immunodeficiency Virus Type 1 (HIV-1) gp120 Elicit Adaptive Immune Responses from Human Cells Engrafted into NOD/SCID Mice and Confer Partial Protection against HIV-1 Challenge J. Virol., February 15, 2005; 79(4): 2124 - 2132. [Abstract] [Full Text] [PDF]
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L. Poluektova, S. Gorantla, J. Faraci, K. Birusingh, H. Dou, and H. E. Gendelman Neuroregulatory Events Follow Adaptive Immune-Mediated Elimination of HIV-1-Infected Macrophages: Studies in a Murine Model of Viral Encephalitis J. Immunol., June 15, 2004; 172(12): 7610 - 7617. [Abstract] [Full Text] [PDF]
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T. Fischer-Smith, S. Croul, A. Adeniyi, K. Rybicka, S. Morgello, K. Khalili, and J. Rappaport Macrophage/Microglial Accumulation and Proliferating Cell Nuclear Antigen Expression in the Central Nervous System in Human Immunodeficiency Virus Encephalopathy Am. J. Pathol., June 1, 2004; 164(6): 2089 - 2099. [Abstract] [Full Text] [PDF]
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M. Moniuszko, C. Brown, R. Pal, E. Tryniszewska, W.-P. Tsai, V. M. Hirsch, and G. Franchini High Frequency of Virus-Specific CD8+ T Cells in the Central Nervous System of Macaques Chronically Infected with Simian Immunodeficiency Virus SIVmac251 J. Virol., November 15, 2003; 77(22): 12346 - 12351. [Abstract] [Full Text] [PDF]
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 Y. Persidsky and H. E. Gendelman Mononuclear phagocyte immunity and the neuropathogenesis of HIV-1 infection J. Leukoc. Biol., November 1, 2003; 74(5): 691 - 701. [Abstract] [Full Text] [PDF]
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 S. Sanchez-Ramon, J. Ma Bellon, S. Resino, C. Canto-Nogues, D. Gurbindo, J.-T. Ramos, and M. Munoz-Fernandez Low Blood CD8+ T-Lymphocytes and High Circulating Monocytes Are Predictors of HIV-1-Associated Progressive Encephalopathy in Children Pediatrics, February 1, 2003; 111(2): e168 - 175. [Abstract] [Full Text] [PDF]
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