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Secretion by Type 2 Predendritic Cells Up-Regulates MHC Class I in the HIV-1-Infected Thymus1
,
*
Gladstone Institute of Virology and Immunology,
Biomedical Sciences Graduate Program, and Departments of
Medicine and
Microbiology and Immunology, University of California, San Francisco, CA 94143
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. IL-3R+ type 2
predendritic cells (preDC2) resident in the thymic medulla secrete
IFN-, which acts on IFN-
R-expressing immature thymocytes to
induce MHC I expression. Furthermore, thymic preDC2 are permissive for
HIV-1 infection and positive for intracellular p24. These data
demonstrate the ability of IFN- secreted by preDC2 to induce MHC I
up-regulation in the HIV-1-infected human thymus. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among the many mechanisms by which HIV-1 subverts the immune system (see Ref. 1 for review), Nef-mediated down-regulation of MHC class I molecules has been proposed as a means by which infected cells can escape detection by MHC I-restricted CD8+ CTLs (2, 3). Nef down-regulates MHC I in a manner dependent on conserved residues in the cytoplasmic tail of HLA-A and -B (4, 5, 6). The specificity of Nef for HLA-A and -B allows HIV-infected cells to evade NK cell attack (7), primarily because NK cells are inhibited by the expression of HLA-C and -E (8).
The above observations are reminiscent of strategies used by other viral pathogens (e.g., adenovirus) to avoid the host immune response (9, 10). However, it remains unclear whether and to what extent Nef-mediated MHC I down-regulation is related to pathologic events as they occur in vivo. In fact, and in apparent contrast to Nef-mediated effects in immortalized cell lines and in isolated PBMC, HIV-1 infection of the human thymus has instead been associated with MHC I up-regulation (11). These experiments, conducted in the human thymus implant of the SCID-hu Thy/Liv mouse, showed distinct up-regulation of MHC I expression on the cell surface of thymocytes. Furthermore, increased MHC I expression correlated with the degree of thymocyte depletion, indicating that MHC I up-regulation is directly related to HIV-1 pathogenesis.
A number of explanations could underlie these discrepant results. First, HIV-1-associated events that occur in thymocytes may be dissimilar from those found in the more mature cells of peripheral lymphoid organs. Second, events that transpire within a hematolymphoid organ such as the thymus may not be reproduced by isolated subpopulations of infected cells maintained in cell culture. Finally, and most trivially, the murine environment of the SCID-hu thymic implant may have unexpected and nonphysiologic consequences on MHC I expression that are unrelated to HIV infection per se.
To explore these findings more completely, we have evaluated the impact
of HIV-1 infection on MHC I expression in isolated subpopulations of
human thymocytes, in human thymic organ culture, and in the SCID-hu
Thy/Liv mouse. Consistent with previous data (12)
demonstrating MHC I down-regulation in thymocytes retrovirally
transduced with Nef, we observed that Nef is associated with modest,
but significant, MHC I down-regulation in productively infected
thymocytes. However, MHC I is dramatically up-regulated on most
(including uninfected) thymocytes from the HIV-1-infected thymus, both
in thymic organ culture and in the SCID-hu Thy/Liv mouse. In these
settings, HIV-1 is observed to interact with type 2 predendritic cells
(preDC2),3 thereby
inducing IFN- secretion and, indirectly, MHC I up-regulation on
IFN-R-positive thymocytes regardless of whether they are HIV-1
infected. These results indicate that the innate immune response
mediates MHC I up-regulation in response to HIV-1 infection of the
thymus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stocks of NL4-3, NL4-3 Xho, and Ba-L were generated as previously described (13). Briefly, high titer NL4-3 (National Institutes of Health AIDS Research and Reference Reagent Program, contributed by M. Martin) and NL4-3 Xho (constructed as described in Ref. 14) stocks were generated by transfection of 293T cells with plasmid DNA. Virus stocks for inoculation of SCID-hu Thy/Liv implants were generated by electroporation of PBMCs with NL4-3 and subsequent culture in PBMC blasts over a 4- to 7-day period. Ba-L (National Institutes of Health AIDS Research and Reference Reagent Program, contributed by S. Gartner, M. Popovic and R. Gallo) stocks were generated in monocyte-derived macrophages. Supernatants were collected after 8 days of culture and were frozen as aliquots. Stocks were analyzed for p24 content and were titrated by limiting dilution assay for 50% tissue culture infectious doses (TCID50) (18).
HIV-1 infection of FTOCs
Thymi were dissected into small (10 mm3)
pieces using sterile knives. Thymic pieces were then transferred
directly into HIV-1 viral stocks (
106
TCID50) or conditioned RPMI 1640 medium from
mock-infected PBMC cultures. Fetal thymic organ cultures (FTOCs) were
inoculated with virus for 4 h at 37°C in a 5%
CO2 incubator. After inoculation, pieces were
transferred to sterile filters (Millipore, Bedford, MA) placed on
Gelfoam (Pharmacia-Upjohn, Kalamazoo, MI) rafts in 700 µl of Yssel’s
medium (Gemini Bio-Products, Calabasas, CA) in 24-well plates.
HIV-1-infected thymic cultures were incubated 7–8 days, and the medium
was changed every 2 days. At the termination of culture, individual
thymus pieces were dispersed and washed before staining for FACS
analysis. To determine whether preDC2 are infected under these
conditions, from HIV-1-infected FTOCs, cultures were rinsed with PBS
with 2% FCS (PBS-FBS) and then digested with 0.4 µg/ml collagenase
and 100 U/ml DNase (Sigma-Aldrich, St. Louis, MO). Digested thymic
tissue was disrupted by repeated pipetting and separated over a
Ficoll gradient (Sigma, Ref. 15). The thymic digest
was then stained for p24, IL3R, and lineage markers (CD3, CD8, CD14,
CD15, CD19, CD20, CD34, CD57) for FACS analysis.
To generate conditioned medium, culture supernatant was removed at the
termination of culture and centrifuged at 250 x g for
5 min at 4°C to remove cellular debris. Supernatant was stored at
-20°C until use. Secondary FTOCs were established using tissue from
an unrelated donor and the conditioned medium from the primary infected
FTOC. Pretreatment of conditioned medium with IFN-, -, or -
neutralizing Abs (BioSource International, Camarillo, CA) was conducted
before establishment of secondary culture. Lamivudine (5 µM; from
National Institutes of Health AIDS Research and Reference Reagent
Program) was added to the culture at the initiation of secondary
cultures. Secondary culture media were changed at 24 h, and
freshly thawed conditioned medium pretreated with the appropriate
neutralizing Ab was added. Stored conditioned media were thawed and
incubated at 37°C for 30 min with saturating amounts of neutralizing
Abs (1000 neutralizing units/ml). Secondary FTOCs were harvested
48 h after the initiation of culture.
HIV-1 infection of SCID-hu Thy/Liv mice
All procedures and practices associated with the use of SCID-hu Thy/Liv mice were approved by the Committee on Human Research or the Committee on Animal Research of the University of California (San Francisco, CA). SCID-hu Thy/Liv mice were generated as previously described (16, 17) and maintained under pathogen-free conditions. Animals in a given cohort were constructed from human fetal tissue from a single donor. Implants were directly inoculated (18) with 50 µl of virus (2000 TCID50) or sterile tissue culture medium. Implants were harvested at the indicated time points, placed into sterile PBS-FCS, and dispersed into a single-cell suspension. Thymocytes were then counted and aliquoted for p24 ELISA, branched DNA assay for HIV-1 RNA, and FACS analysis as previously described (19).
FACS staining and analysis
Dispersed thymocytes from FTOC cultures and SCID-hu Thy/Liv implants were washed, pelleted, and resuspended in 50 µl of mAbs diluted in 1 mg/ml human gamma globulin and incubated on ice for 30 min. After incubation, cells were rinsed, pelleted, and resuspended in 200 µl of PBS-FCS for immediate FACS analysis. Intracellular staining was performed as follows. After initial staining of surface cell markers, cells were rinsed and pelleted. Pellets were resuspended in 200 µl of a solution of 1% paraformaldehyde, 1 mg/ml human gamma globulin, and 0.1% Tween 20 in PBS-FCS. Cells were incubated at room temperature for 1 h in the dark and then rinsed twice and pelleted. Pellets were resuspended in Ab to intracellular p24 diluted in PBS-FCS and stained on ice for 30 min. After incubation, cells were rinsed, pelleted, and resuspended in PBS-FCS for immediate FACS analysis.
Four-color FACS staining was performed with the indicated combinations of the following Abs: KC57 (anti-p24) FITC, CD14-allophycocyanin, and CD56-allophycocyanin (all from Coulter, Hialeah, FL); CD3-FITC, CD8-PerCP, CD123-PE, CXCR4-allophycocyanin, CCR5-allophycocyanin, and CD34-allophycocyanin (all from BD Biosciences, San Jose, CA); anti-HLA-A, -B, -C (pan-MHC I; W6/32)-PE (DAKO, Carpinteria, CA); HLA-A2 PE (One Lambda, Canoga Park, CA; PE-conjugated in our laboratory by C. King); and CD118-PE, CD4-allophycocyanin, CD8-allophycocyanin, CD14-allophycocyanin, CD19-allophycocyanin, and CD20-allophycocyanin (all from Caltag Laboratories, Burlingame, CA).
ELISA
Conditioned media from HIV-1-infected FTOCs were harvested 7
days after inoculation and analyzed using IFN-, IFN-, or IFN-
ELISA kits (BioSource International). Importantly, the IFN- ELISA
employed in these studies detected human IFN-A, 2, A/D, D,
K, and 4b.
Immunohistochemistry
HIV-1-infected SCID-hu Thy/Liv implants were harvested at
designated time points and snap-frozen by immersing tissue suspended in
Tissue Freezing Medium (Fisher, Pittsburgh, PA) in isopentane cooled in
dry ice. Eight-micrometer sections were cut on a Shandon microtome
(Thermo Shandon, Pittsburgh, PA), dried overnight, fixed in a 1/1
acetone/methanol solution, and dried for 30 min at room temperature.
Endogenous peroxidase was neutralized by pretreatment with 0.3%
H2O2 in 0.3% horse serum
in TBS. Sections were then blocked with 10% horse serum in 0.1%
saponin-TBS. Primary Ab incubations were conducted at 37°C in 5%
CO2 for 1 h as previously reported for
IFN- (20). Secondary Ab incubations were performed at
room temperature with biotin-conjugated anti-mouse Abs (Vector
Laboratories, Burlingame, CA). Sections were developed with ABC Elite
and diaminobenzidene reagents (Vector Laboratories) and mounted using
Crystal Mount (Fisher).
IFN- treatment of HIV-1-infected FTOCs
FTOCs were plated on Gelfoam rafts in 24-well plates and were
pretreated overnight with 1000 U/ml IFN- (Schering-Plough,
Kenilworth, NJ). The next day, FTOCs were inoculated with HIV-1 as
described above. HIV-1-infected FTOCs were plated onto Gelfoam rafts,
and IFN- (1000 U/ml) was added at the indicated times after
inoculation. Seven days after inoculation FTOCs were harvested and
dispersed by repeated pipetting. A portion of the cells were reserved
for p24 ELISA analysis, and the remaining cells were stained for FACS
analysis with p24-FITC, MHC I-PE, or HLA-A2 PE, CD8-PerCP, and
CD4-allophycocyanin.
Assessment of HIV-1 gag DNA in preDC2
Lineage (CD3, CD8, CD14, CD15, CD16, CD19, CD20, CD34,
CD57)-negative,
IL-3R+CD11c- preDC2s
and CD3+ thymocytes were isolated by sorting on a
FACSVantage (BD Biosciences) from FTOCs 7 days after inoculation with
NL4-3 or Ba-L. After sorting, cells were pelleted and lysed, and
lysates were used as templates for real-time PCR. Mock-infected FTOCs
were consistently negative for gag expression. The
primers used were as follows: -globin antisense,
5'-CCTGGGAGTAGATTGGCCAA; -globin sense,
5'-GAAGAGCCAAGGACAGGTACG; gag antisense,
5'-TGCTATGTCACTTCCCCTTGG; and gag sense,
5'-CCATCAATGAGGAAGCTGCA. The probes used were as follows:
gag, 5'-FAM-TGCATGCACTGGATGCAATCTATCCCATT-TAMRA; and
-globin, 5'-FAMCTGTCATCACTTAGACCTCACCCTGTG-TAMRA. PCR
were run on an ABI PRISM 7700 sequence detector (Applied Biosystems,
Foster City, CA) using the following parameters: 10 min at 95°C and
then 40 cycles of 15 s at 95°C and 60 s at 58°C. Primers
and probe pairs were designed using PerkinElmer PrimerExpress
software, and data were analyzed using Sequence Detector version
1.6.3 software (both from PerkinElmer, Boston, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous experiments have shown that MHC I is up-regulated on
thymocytes from HIV-1-infected thymus (11). FTOCs were
infected with Nef+ (NL4-3) (21) or
Nef- (NL4-3 Xho) (14)
CXCR4(X4)-using HIV-1 isolates. Cultures were harvested 7 days after
virus inoculation, and relative levels of infection and MHC I
expression were determined by flow cytometry. Compared with thymocytes
harvested from mock-infected FTOCs, MHC I expression on thymocytes, as
assessed by staining with a pan-MHC I Ab, was increased >10-fold on
the majority of thymocytes after infection with NL4-3 or NL4-3 Xho
(Fig. 1
, A and B).
MHC I up-regulation was observed on both p24+ and
p24- thymocytes (Fig. 1A), indicating
that MHC I up-regulation was independent of productive HIV-1 infection.
Although MHC I up-regulation was observed on most thymocytes,
productively infected thymocytes from NL4-3-infected cultures
consistently showed significant lowered MHC I expression compared with
p24+ thymocytes from NL4-3 Xho-infected cultures
(Fig. 1B), indicating that Nef-mediated MHC I
down-regulation can occur to a certain degree within infected cells in
the HIV-1 infected thymus. As Nef has been reported to primarily affect
HLA-A and -B expression (4), thymocytes from mock-infected
and NL4-3-infected thymi were assessed for down-regulation of HLA-A2.
As shown in Fig. 1C, HLA-A2 down-regulation occurs in
NL4-3-infected thymocytes in a manner similar to that observed with
pan-MHC I Ab, with lower levels of expression in
p24+ compared with p24-
cells. Yet, the expression of HLA-A2 on p24+
cells was still significantly higher than that found on cells from
mock-infected cultures (Fig. 1, A and D). Similar
results were obtained in infections of SCID-hu Thy/Liv implants (data
not shown). These data indicate that HIV-1 infection in the intact
thymus induces significant up-regulation of MHC I on both infected and
uninfected cells.
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In time-course experiments conducted in SCID-hu Thy/Liv mice, MHC
I up-regulation was observed as early as 4 days after inoculation with
NL4-3 (data not shown). After inoculation with X4- or CCR5 (R5)-using
HIV-1, NL4-3 (Fig. 2A) and
Ba-L (Fig. 2B), respectively, MHC I up-regulation on
CD4+CD8+ thymocytes
strongly correlated (p 
0.0001) with markers
of productive HIV-1 infection, e.g., HIV-1 p24 protein and RNA (Fig. 2, C–F). Therefore, up-regulation of MHC I is a
direct result of HIV-1 infection of the human thymus and correlates
with the degree of productive HIV-1 infection. These data support the
idea that MHC I up-regulation correlates with HIV-1 pathogenesis in the
thymus, as previously reported (11).
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mediates MHC I up-regulation in HIV-1-infected thymus
To explore the possibility that a soluble factor is responsible
for up-regulation of MHC I on uninfected
CD4+CD8+ double-positive
(DP) thymocytes in the HIV-1-infected thymus, we tested whether
conditioned medium from HIV-1-infected FTOCs could lead to MHC I
up-regulation on thymocytes. Conditioned medium from HIV-1-infected or
mock-infected cultures was transferred to freshly plated FTOCs from a
different donor in the presence of the nucleoside analog lamivudine (to
inhibit infection of the secondary cultures by transferred virus).
After 2 days in culture, the level of MHC I expression on DP thymocytes
was similar to that observed after primary infection of FTOC (Fig. 3A), implicating the
involvement of a soluble factor.
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(Fig. 3
B), but not IFN-
or - (data not shown), were detected by ELISA in all conditioned
medium samples from FTOCs productively infected with either NL4-3 or
Ba-L. The level of IFN- in the conditioned media ranged from 1000 to
1500 pg/ml (300–500 IU/ml) and was proportional to the level of MHC I
expression observed in the primary culture (data not shown).
Importantly, transfer of cultured medium up-regulated MHC I on
thymocytes with kinetics similar to administration of exogenous IFN-
(data not shown).
To determine whether the presence of IFN- is responsible for the MHC
I up-regulation observed in HIV-1 infection of the thymus, neutralizing
Abs against IFN-, -, and - were added to the conditioned media
before transfer to secondary cultures. Up-regulation of MHC I
expression was blocked by pretreatment of conditioned media from NL4-3-
or Ba-L-infected FTOC with neutralizing Abs against IFN-;
pretreatment with neutralizing Abs against either IFN- or IFN-
had no effect (Fig. 3C). These data demonstrate that IFN-
acts as a soluble mediator of MHC I up-regulation on DP thymocytes in
both X4- and R5-using HIV-1 infection of the human thymus.
To determine whether DP thymocytes are capable of responding to
IFN-, expression levels of the IFN-R (CD118) were evaluated on
various subpopulations of thymocytes (Table I). A gradient of receptor expression was
found, with highest levels on early maturational intermediates
(intrathymic T progenitor and DP) and lower levels on more
mature CD3+ cells that express either CD4 or CD8
(SP4 and SP8 thymocytes, respectively). These levels corresponded to
the degree of MHC I up-regulation observed on each subpopulation 40–46
days after inoculation with Ba-L. Interestingly, very little
IFN-R expression was found on CD3+
peripheral blood T cells, indicating that these cells may not be
responsive to IFN-.
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+ cells secrete IFN- in response to HIV-1
infection of the human thymus
A subset of immature dendritic cells (known as preDC2,
plasmacytoid dendritic cells, or natural IFN-producing cells) has been
shown to secrete large amounts of IFN- upon incubation with a wide
range of viruses (22, 23). PreDC2 have been described in
the peripheral blood, but also reside in the thymus (24, 25). To determine whether IL-3R
(CD123+) preDC2 might be responsible for the
production of IFN- in HIV-1-infected thymic tissue,
immunohistochemical stains were used to detect IL-3R and IFN- in
mock- and HIV-1-infected SCID-hu Thy/Liv implants. In mock-infected
animals, large granular IL-3R+ cells were
observed in the medulla (Fig. 4A), but these cells did not
have detectable levels of IFN- (Fig. 4B). Twenty-one days
after NL4-3 infection, analysis of serial sections indicated that most
of the IL-3R+ cells were also
IFN-+ (arrows in Fig. 4, C and
D); in all cases, the positively staining cells were large,
granular, and localized to the thymic medulla. Similar results were
obtained in Ba-L-infected animals 42 days after HIV-1 inoculation (data
not shown). These data demonstrate that preDC2-like cells resident in
the uninfected thymus secrete IFN- after HIV-1 infection.
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+ preDC2 isolated from the
human thymus were found to express both CCR5 and CXCR4 (Fig. 4E) as well as CD4 (24, 25), indicating that
they could be permissive for HIV-1 infection. Indeed, thymic preDC2
from HIV-1-infected FTOCs were found to be positive for intracellular
p24 by FACS analysis (Fig. 4F). Furthermore, we found
significant HIV-1 DNA in sorted preDC2 from both NL4-3-infected (265
gag copies/1000 sorted cell equivalents; n =
4) and Ba-L-infected (940 gag copies/1000 sorted cell
equivalents; n = 4) FTOCs by real-time PCR. These data
demonstrate that preDC2 are infected by HIV-1 in the thymus and that
both CXCR4- and CCR5-using HIV-1 isolates can infect thymic preDC2.
Further study will be necessary to determine what impact HIV-1
infection has on preDC2 function and IFN- secretion.
Early treatment of FTOCs with IFN- neutralizes HIV infection
IFN- has long been recognized for its potent antiviral effects
(26, 27). IFN- induces resistance to viral replication
in cells, increases the expression of MHC I molecules, and activates NK
cells to suppress viral replication in many systems. As shown in Table I, some thymocyte subpopulations express high levels of the IFN-R
and may be able to resist viral infection after IFN--mediated
activation of intracellular antiviral cascades.
To determine the ability of IFN- secretion to block HIV-1 infection,
we treated FTOCs with IFN- at various times after HIV-1 inoculation.
IFN- treatment was found to block productive HIV-1 infection, as
measured by cell-associated p24 production and intracellular staining
for p24 on day 7 (Fig. 5). There was no
significant advantage to pretreatment of FTOCs with IFN-, as
initiation of treatment up to 4 days after viral inoculation
significantly inhibited HIV-1 replication assessed on day 7. These data
demonstrate that IFN-, if produced early in infection and in
sufficient quantities, is capable of blocking productive HIV-1
infection and/or spread.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion by preDC2. Such up-regulation occurs even though Nef
continues to down-regulate MHC molecules to a certain degree on the
surface of infected thymocytes. HIV-1-induced MHC I up-regulation can
be completely blocked by addition of neutralizing Abs to IFN-.
Furthermore, IFN- secretion is tied to productive viral infection,
as MHC I up-regulation correlates strongly with markers of active viral
replication, such as viral RNA and p24 production. Although IFN- is
protective against viral infection, IFN- secretion by preDC2 in
HIV-1-infected thymus does not block thymocyte depletion. This
indicates that there is an innate immune response to HIV-1 infection in
the thymus, but it cannot adequately control HIV-1-induced
pathogenesis. In addition, we observed a significant Nef-dependent
down-regulation of HLA-A and -B molecules on the surface of
productively infected thymocytes. Although we demonstrated that
administration of exogenous IFN- is protective against productive
viral replication, it is clear from our studies that endogenous
production of IFN- by itself is insufficient to impede HIV-1
pathogenesis in the thymus.
The ability to modulate MHC I expression is a common viral immune
evasion tactic. Evidence of Nef-mediated down-regulation of MHC I in in
vitro systems has led to the hypothesis that Nef may allow
HIV-1-infected cells to evade cytotoxic T cell clones specific for
viral Ags (2, 3). Our data show that Nef down-regulates
MHC I expression on a population of p24+
thymocytes. These observations demonstrate that HIV-1-associated
events, specifically Nef-mediated down-regulation of MHC I, are similar
in thymocytes and more mature peripheral cells. There is a lower
relative down-regulation of MHC I in infected thymocytes compared to
reports using other cell types (2, 3), which is probably
due to the relatively low MHC I expression on thymocytes
(28). However, it is clear from our data that the dominant
effect in intact HIV-1-infected thymus is IFN--induced MHC I
up-regulation on both HIV-1-infected and uninfected thymocytes. These
experiments underscore the possibility that virus-mediated effects are
substantially different in intact lymphoid organs than in dispersed
cell culture because of the rich network of accessory cells that
mediate important events in the innate immune response. Finally, we
demonstrate that we can recapitulate responses identical with HIV-1
infection of FTOCs in SCID-hu thymic implants, suggesting that the
observations of MHC I up-regulation in this system are physiologically
relevant.
Recent data have led to the speculation that preDC2 act as an important
link between the innate and adaptive immune systems (29).
It has been shown previously that preDC2 secrete IFN- when cultured
in vitro with HIV-1 (20, 30). This is the first
demonstration of in vivo IFN- secretion by preDC2 in response to
viral stimulation. In vitro experiments have shown that maturation of
preDC2 after initial response to virus results in the secretion of
cytokines and the initiation of an adaptive immune response
(29). Additional experiments will be necessary to
determine whether preDC2 can perform these functions in the context of
thymic viral infection. We additionally demonstrate that thymic preDC2
express the CXCR4 and CCR5 coreceptors and are productively infected by
HIV-1. This is the first demonstration that preDC2 are infected by
HIV-1 in vivo; additional experiments will be necessary to determine
whether preDC2 from the peripheral blood and lymphoid tissue of
HIV-1-infected patients are similarly infected. It will also be
interesting to determine the effect of HIV-1 infection of preDC2 on
continued antiviral responses by these cells.
The role of IFN- in this dynamic system is still somewhat unclear.
Exogenous IFN- acts as a potent antiviral when added to
HIV-1-infected cultures up to 4 days after initial inoculation. Our
attempts to block endogenous IFN- production with neutralizing Abs
in FTOCs have been unsuccessful, most likely because of the close
proximity of IFN--producing cells and IFN-R-bearing cells.
Since IFN- is secreted after HIV-1 infection and has been shown in
model systems to be capable of suppressing viral replication, the
question arises as to why endogenous IFN- secretion by preDC2 is not
more effective at containing HIV-1 replication. MHC I up-regulation, a
direct marker of IFN- secretion by preDC2, instead acts as a direct
correlate of viral replication. This dichotomy may reflect a compromise
in preDC2 function after HIV-1 infection. It additionally may reflect
inadequate or delayed IFN- secretion by preDC2 and/or negative
effects of IFN- secretion on thymopoiesis.
IFN--induced MHC I up-regulation may have detrimental effects on
thymopoiesis by disrupting the normal selection milieu in
HIV-1-infected thymus. Thymic selection is critically dependent on the
avidity of the MHC I and the TCR-CD8 coreceptor signaling complex. We
observed significant increases in expression of MHC I on the surface of
thymocytes and thymic epithelial cells (data not shown), which may
negatively impact the avidity interactions critical to the selection of
developing thymocytes. Studies using high-avidity murine models of T
cell selection have shown that mature selected thymocytes displayed
decreased CD8 cell surface density (31, 32). Decreased
cell surface expression of CD8 presumably lowers the overall affinity
of the TCR-CD8 signaling complex for its high avidity MHC I ligand,
allowing thymocytes to escape negative selection. We observed a similar
down-regulation of CD8 on the surface of mature
CD3+CD8+ thymocytes
selected in the HIV-1-infected thymus (data not shown), suggesting that
MHC I up-regulation may have detrimental effects on thymocyte
maturation.
HIV-1 demonstrates an enormous capacity to evade immune system
surveillance. We demonstrate here that HIV-1 infection of the human
thymus induces MHC I up-regulation on IFN-R-bearing thymocytes as
a result of secretion of IFN- by preDC2. IFN- secretion
up-regulates MHC I on infected and uninfected thymocytes, even though
Nef-dependent MHC I down-regulation can still be observed to a certain
degree on infected cells. Importantly, IFN- secretion is not
sufficient to inhibit viral pathogenesis. This is particularly
interesting in light of data demonstrating that exogenous IFN-
blocks viral p24 production. These data indicate that there may be an
additional level of complexity to the interactions between HIV-1 and
sentinel cells such as preDC2 that reside in primary lymphoid tissue.
They also underscore the ability of the immune response, at least in
primary lymphoid tissue, to counteract viral immune evasion
mechanisms.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Joseph M. McCune, Gladstone Institute of Virology and Immunology, University of California, P.O. Box 419100, San Francisco, CA 94141-9100. E-mail address: mmccune{at}gladstone.ucsf.edu
3 Abbreviations used in this paper: preDC2, type-2 predendritic cell; TCID50, 50% tissue culture infectious dose; FTOC, fetal thymic organ culture; DP, double-positive.
Received for publication September 5, 2001. Accepted for publication October 29, 2001.
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M. Schweneker, D. Favre, J. N. Martin, S. G. Deeks, and J. M. McCune HIV-Induced Changes in T Cell Signaling Pathways J. Immunol., May 15, 2008; 180(10): 6490 - 6500. [Abstract] [Full Text] [PDF]
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 H. Schmidlin, W. Dontje, F. Groot, S. J. Ligthart, A. D. Colantonio, M. E. Oud, E. J. Schilder-Tol, M. Spaargaren, H. Spits, C. H. Uittenbogaart, et al. Stimulated plasmacytoid dendritic cells impair human T-cell development Blood, December 1, 2006; 108(12): 3792 - 3800. [Abstract] [Full Text] [PDF]
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H. Donaghy, J. Wilkinson, and A. L. Cunningham HIV interactions with dendritic cells: has our focus been too narrow? J. Leukoc. Biol., November 1, 2006; 80(5): 1001 - 1012. [Abstract] [Full Text] [PDF]
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S. K. Choudhary, N. R. Choudhary, K. C. Kimbrell, J. Colasanti, A. Ziogas, D. Kwa, H. Schuitemaker, and D. Camerini R5 Human Immunodeficiency Virus Type 1 Infection of Fetal Thymic Organ Culture Induces Cytokine and CCR5 Expression J. Virol., January 1, 2005; 79(1): 458 - 471. [Abstract] [Full Text] [PDF]
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K. B. Gurney, A. D. Colantonio, B. Blom, H. Spits, and C. H. Uittenbogaart Endogenous IFN-{alpha} Production by Plasmacytoid Dendritic Cells Exerts an Antiviral Effect on Thymic HIV-1 Infection J. Immunol., December 15, 2004; 173(12): 7269 - 7276. [Abstract] [Full Text] [PDF]
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T. Ueno, H. Tomiyama, M. Fujiwara, S. Oka, and M. Takiguchi Functionally Impaired HIV-Specific CD8 T Cells Show High Affinity TCR-Ligand Interactions J. Immunol., November 1, 2004; 173(9): 5451 - 5457. [Abstract] [Full Text] [PDF]
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F. Brilot, V. Geenen, D. Hober, and C. A. Stoddart Coxsackievirus B4 Infection of Human Fetal Thymus Cells J. Virol., September 15, 2004; 78(18): 9854 - 9861. [Abstract] [Full Text] [PDF]
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S. Turville, J. Wilkinson, P. Cameron, J. Dable, and A. L. Cunningham The role of dendritic cell C-type lectin receptors in HIV pathogenesis J. Leukoc. Biol., November 1, 2003; 74(5): 710 - 718. [Abstract] [Full Text] [PDF]
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M. E. Keir, M. G. Rosenberg, J. K. Sandberg, K. A. Jordan, A. Wiznia, D. F. Nixon, C. A. Stoddart, and J. M. McCune Generation of CD3+CD8low Thymocytes in the HIV Type 1-Infected Thymus J. Immunol., September 1, 2002; 169(5): 2788 - 2796. [Abstract] [Full Text] [PDF]
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K. Abel, M. J. Alegria-Hartman, K. Rothaeusler, M. Marthas, and C. J. Miller The Relationship between Simian Immunodeficiency Virus RNA Levels and the mRNA Levels of Alpha/Beta Interferons (IFN-{alpha}/{beta}) and IFN-{alpha}/{beta}-Inducible Mx in Lymphoid Tissues of Rhesus Macaques during Acute and Chronic Infection J. Virol., July 17, 2002; 76(16): 8433 - 8445. [Abstract] [Full Text] [PDF]
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L. Pedroza-Martins, W. J. Boscardin, D. J. Anisman-Posner, D. Schols, Y. J. Bryson, and C. H. Uittenbogaart Impact of Cytokines on Replication in the Thymus of Primary Human Immunodeficiency Virus Type 1 Isolates from Infants J. Virol., June 14, 2002; 76(14): 6929 - 6943. [Abstract] [Full Text] [PDF]
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P.-O. Vidalain, D. Laine, Y. Zaffran, O. Azocar, C. Servet-Delprat, T. F. Wild, C. Rabourdin-Combe, and H. Valentin Interferons Mediate Terminal Differentiation of Human Cortical Thymic Epithelial Cells J. Virol., June 5, 2002; 76(13): 6415 - 6424. [Abstract] [Full Text] [PDF]
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) or mock-infected control (bold line) SCID-hu Thy/Liv implants 21
days after inoculation. B, Representative staining of
MHC I on DP thymocytes from Ba-L-infected (
) and
demonstrate a clear positive correlation between indicators of viral
replication (p24 and HIV-1 RNA) and MHC I expression on DP
thymocytes.
