 |
Introduction |
CD8+ T lymphocytes play a protective role during the
acute and chronic phases of HIV-1 infection. In addition to eliminating productively infected cells through their
cytotoxic activity, CD8+ T cells also release soluble factors
(macrophage inflammatory protein [MIP]-1
, MIP-1
,
regulated on activation, normal T cell expressed and secreted [RANTES], IL-16, and other unidentified factors)
that inhibit cell entry and intracellular replication of HIV-1
(for review see references 1 and 2). In most adult patients,
HIV triggers a rapid and strong cytotoxic CD8+ T cell response that appears to limit viral replication during the initial acute phase of the infection (3, 4). The efficiency of the
initial CD8+ T cell response, as reflected by the viral burden at the end of the acute episode, is thought to determine the rate of disease progression to AIDS (5, 6). In the
majority of adults, the viral load declines rapidly after primary HIV infection and the disease progresses slowly, with
a median clinical latent phase of about 9 yr. In contrast, the
evolution of neonatal HIV infection, resulting from maternal-infant transmission, is often much faster, with the highest incidence of pediatric AIDS occurring during the first
year of life (7). In perinatally infected infants, plasma HIV
levels peak at 1 to 2 mo of age and decline only very slowly
during the first 2 yr of life (8). This may be related to an insufficient neonatal HIV-specific CD8+ T cell response
characterized by a smaller amplitude and a more restricted
TCR repertoire than in adults (9). Here we provide a
mechanism that may contribute to the inefficient response
of neonatal CD8+ T cells by showing that these cells can be
productively infected in vitro by HIV.
 |
Materials and Methods |
Lymphocyte Preparation and Culture Conditions.
CD8+ and CD4+
T cell subsets were isolated from heparinized umbilical cord
blood as described (10). In brief, mononuclear cells obtained by
centrifugation on Ficoll-Metrizoate gradients were treated with
L-leucine methyl ester to remove monocytes and NK cells; enriched T cells were isolated by treating cells forming rosette with
SRBCs by means of Lympho-Kwik T (One Lambda, Canogalark, CA). CD8+ T cells were positively selected with Dynabeads M-450 CD8 (Dynal, Great Neck, NY) and further depleted of CD4+ cells with Dynabeads M-450 CD4; CD4+ T cells
were obtained by treating enriched T cells with Lympho-Kwik T
helper (One Lambda). Neonatal T cell subsets were >98% CD3+
and <1% CD14, CD20, CD56, or CD16 positive, respectively;
the CD8+ subset was >95% TCR-
/
+ CD8-
/
+, >99%
CD45RA+/RO
, and contained no detectable CD4 or CD29
positive cells. T cells were submitted to one or two cycles of activation and IL-2 expansion as described (11). At each cycle, T
cells were activated with anti-CD3 mAb (UCHT-1, 200 ng/ml;
a gift from P. Beverley, University College and Middlesex School
of Medicine, London, UK) and irradiated CD32, B7.1 transfected L cells for 3 d, and cells were then washed twice and expanded in fresh medium supplemented with 50 IU/ml of IL-2 for
4 d. T cells were then washed and either stored in liquid nitrogen, until use for HIV infection, or subjected to another cycle of
activation/expansion.
In some experiments, CD8+ T cells (5 × 105 cells/ml) were
stimulated with irradiated allogeneic dendritic cells (5 × 104
cells/ml) in the presence of IL-2 (50 IU/ml). Dendritic cells were
derived from adult blood monocytes cultured for 9 d with IL-4,
GM-CSF, and TNF-
exactly as described (12).
Flow Cytometry.
One- or two-color flow cytometric analysis
was performed on a FACSort® (Becton Dickinson, Montreal,
Canada) according to standard procedures. The following FITC-
or PE-labeled mAbs or isotype-matched mouse Ig were purchased from Becton Dickinson: anti-CD8-
(Leu 2a), anti-CD4
(Leu 3a), anti-CD3, anti-CD20, anti-CD14, anti-CD56, and anti-CD16. CD4 was also stained with OKT4 (American Type Culture Collection, Rockville, MD); mAb to CD8-
/
heterodimer
(2ST8) was given by E. Reinherz (Harvard Medical School, Boston, MA); the anti-CXCR4/fusin mAb (12G5) has been described (13).
Reverse Transcriptase PCR.
Total cellular RNA was extracted
from resting or activated cord blood CD4+ or CD8+ T cells using RNA ease Total RNA Kit (Qiagen, Chasworth, CA) and pretreated with 10 U/ml RNAse-free DNAse (GIBCO BRL, Gaithersburg, MD) before amplification. Synthesis of cDNA and ensuing PCR amplification were performed using TitanTM reverse
transcriptase (RT)-PCR kit (Boehringer Mannheim, Indianapolis, IN) according to the supplier's instruction manual. PCR products were separated on a 1% agarose gel and stained with ethidium
bromide. Chinese hamster ovary cell RNA served as negative control for CD4, CXCR4, and CCR5 genes; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was amplified to confirm equal
efficiency of amplification of each RNA. No PCR product was
detected in control amplification containing no added RNA, Taq,
or RT DNA polymerase. Primers for amplification were as follows:
CXCR4: 5'-AGAATTCAACCAGCGGTTACCATGGAGGGGATC, 5'-AGAATTCGTCTTTTACATCTGTGTTAGCTGGAG; CCR5: 5'-TCGATCCGGTGGAACAAGATGGATTATCAAG, 5'-TCGGATCCAAGCCATGTGCACAACTCTGACTG; and CD4: 5'-GCAGTGGCGAGCTGTGGT, 5'-GGGTCCCCACACCTCACAGG.
HIV Infection and Quantitative Gag PCR.
Either freshly activated or cryopreserved neonatal CD4+ and CD8+ cells were infected with HIV-1US-1 or HIV-1NL4-3 as previously described (14,
15). In brief, for each infection 5 × 106 cells were centrifuged,
washed, and resuspended in 400 µl of media containing 1-3 x 104
tissue culture and infectious dose (TCID)50 of HIV-1. The cells were incubated at 37°C for 2 h, washed three times to remove
excess virus, and resuspended in 50% conditioned media at 106/ml.
At t = 0, 2, 72, and 144 h after infection, 1,000,000 cells were
pelleted and frozen at
70°C. The cell pellets were lysed, amplified by PCR using HIV gag-specific primers, and the amplified sequences were detected by hybridization to a radiolabeled oligonucleotide specific for internal gag sequences. The hybridized
products were separated by gel electrophoresis and exposed to a
PhosphorImager screen for 1 h. To ensure that the reactions were
performed within the linear range of the assay, log increments
HIV gag plasmid standards were amplified at the same time. To
show that equivalent levels of input DNA were present in each
PCR reaction, human
-globulin sequences were PCR amplified
as described (15).
Intracellular HIV Gag-pol Staining.
After washes 1-2 × 106
cells were stained with optimized concentrations of cell surface
marker antibodies for 1 h at 4°C. The cells were washed twice
with sterile PBS, pH 7.4, and fixed in 100 µl of PermeaFix
(Ortho Diagnostics, Raritan, NJ) for at least 1 h at room temperature. Cells were washed in sterile PBS, pH 7.4, pelleted by centrifugation, and then washed again in 2× standard saline citrate
(SSC). After centrifugation, the cell pellet was resuspended in hybridization solution (2× SSC, 30% formamide, sonicated salmon
sperm, yeast transfer RNA) containing 500 ng of 5-carboxy-fluorescein double-end-labeled, gag-pol-specific oligonucleotide probes
or gag-pol sense oligonucleotides as a negative control probe
cocktail (16). The intracellular hybridization was performed at
42°C for 1 h followed by successive washes in 2× SSC, 0.5% Triton X-100, and 1× SSC, 0.5% Triton X-100 at 42°C. The cells
were resuspended for analysis in PBS, pH 8.3, and analyzed on a
flow cytometer (Epic XL; Coulter, Miami, FL).
 |
Results and Discussion |
Highly purified umbilical cord blood CD8+ T cells
(>98% CD3+, >95% TCR-
/
+, CD8-
/
+, no detectable CD4+ cells) and CD4+ T cells (>98% CD3+
CD4bright, no detectable CD8+ cells) were activated with
anti-CD3 mAb cross-linked on CD32 and B7.1 transfected
L cells, expanded in IL-2-supplemented medium, and then
infected with the macrophage-tropic HIVUS-1 or with the T cell-tropic (T-tropic) HIVNL4-3 virus; HIV replication
was evaluated at days 3 and 6 after infection by quantitative
PCR analysis of gag DNA accumulation. Both CD8+ and
CD4+ T cell subsets supported significant replication of the
macrophage tropic (M-tropic), but not the T-tropic, strain
of HIV (Fig. 1), although the initial infection was slower in
CD8+ than in CD4+ T cells, as judged from the day 3 values. That HIV infected CD8+ T cells, and not contaminating CD4+ cells was supported by 3 lines of evidence. First,
neonatal CD8+ T cells were directly shown to contain intracellular HIV. After infection with HIVUS-1 for 6 d,
CD8+ T cells were surface stained for CD8 expression and
stained intracellularly for HIV gag and pol RNA sequences.
Fig. 2 shows that only CD8+ T cells contained HIV-1 sequences. In addition, to confirm that a spreading infection
was occurring in the neonatal CD8+ T cells, samples were
collected on both days 3 and 6 after infection and analyzed.
At day 3, 3.4% of the cells were double positive for CD8
and HIV RNA, and by day 6 the number grew to 12.3%,
which is consistent with a productive infection (data not
shown). Second, CD8+ T cell cultures did not contain detectable CD4 single positive cells at day 6 after infection
(data not shown). Third, CD4 Ag and CD4 messenger
RNA (mRNA) were undetectable in freshly prepared CD8+ T cells, as assessed by flow cytometry and RT-PCR
analysis respectively (Fig. 3, A and B). Thus, activated neonatal CD8+ T cells can be productively infected by primary
M-tropic HIV isolates, responsible for disease transmission,
but not by T-tropic HIV. The apparent resistance of neonatal CD4+ and CD8+ T cell subsets to T-tropic HIV
could be related to the recent finding that anti-CD3/B7-
activated naive CD4+ T cells isolated from adult blood are
also resistant to T-tropic HIV, as a result of postentry block
in viral replication (17).

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Fig. 1.
Infection of neonatal CD4+ and CD8+ cells. Umbilical cord
blood CD4+ and CD8+ T cells were activated with anti-CD3 and B7-1-transfected CD32 L cells for 3 d, washed, expanded in IL-2, and infected with either a CCR5-dependent virus, HIVUS1, or a CXCR4-dependent virus, HIV-1NL4-3. The PhosphorImage shows the accumulation of
gag DNA in CD4 and CD8 cultures at 0, 2, 72, and 144 h after infection.
To ensure equivalent amounts of DNA were in the PCR reaction, human -globulin sequences were amplified.
|
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Fig. 2.
Intracellular HIV-1 staining of neonatal CD8+ cells. Neonatal CD8+ T cells were harvested 6 d after infection and stained with both
anti-CD8 and labeled HIV DNA probes. Left, FACS® analysis of cells infected with HIVUS1; right, a mock-infected control.
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Fig. 3.
Expression of CD4, CXCR4, and CCR5 on neonatal CD8+ T cells. Umbilical cord blood CD8+ T cells were activated with anti-CD3
mAb immobilized on B7.1 and CD32-transfected L cells and expanded in IL-2-supplemented medium. (A) Two-dimensional contour plot showing expression of CD4 and CD8 before T cell activation, after 24 and 72 h of primary activation, and at the end of a second cycle of activation/IL-2 expansion
(day 14). Similar results were obtained by staining with OKT4 mAb, recognizing another CD4 epitope than Leu3a mAb. More than 95% of resting or
activated CD8+ T cells were stained brightly with 2ST8 mAb (received from E. Reinherz, Harvard Medical School, Boston, MA) specific for the CD8- / heterodimer. (B) Expression of CD4, CXCR4, and CCR5 mRNA in resting and activated (day 7) neonatal CD4+ and CD8+ T cells. (C) Comparison of CD4 expression on activated CD4+ and CD8+ T cells. Cells were stained with anti-CD4 mAb at the end of the first cycle of activation/IL-2
expansion (day 7). (D) CD4 expression on CD8+ T cells activated with either plate-coated anti-CD3 (10 µg/ml), immobilized anti-CD3 together with
soluble anti-CD28 (clone 9.3, 500 ng/ml), anti-CD3 (200 ng/ml) immobilized on B7.1 CD32 L cells, or allogeneic dendritic cells together with IL-2 (50 IU/ml). Cells were stained at day 7 with anti-CD4 and anti-CD8 mAbs. (E) CXCR4 expression on activated CD8+ T cells. Anti-CD3/B7.1-activated
CD8+ T cells were stained with 12G5 mAb at day 7.
|
|
To be susceptible to HIV infection, a cell must express
CD4, the primary HIV receptor, together with either
CCR5 (the coreceptor for M-tropic strains), or CXCR-4/
fusin (the coreceptor for T-tropic strains) (for review see
reference 21). According to this paradigm, activated neonatal CD8+ T cells should express CD4 together with at least
CCR5. Indeed, we found that neonatal single positive
CD8+ T cells started to express low to moderate levels of
CD4 within 24 h after anti-CD3/B7.1 activation (Fig. 3
A); CD4 expression reached a plateau at 72 h (60 to >90%
double positive cells, n = 24) and remained stable for at
least 2 wk (the duration of the present experiments). Flow
cytometric analysis of anti-CD3/B7.1-stimulated CD8+ T
cells stained with anti-CD4 revealed a unimodal histogram, suggesting that the ability to coexpress CD4 is a common
property of most, if not the entire, neonatal CD8+ T cell
population (Fig. 3 C). Quantitatively, CD4 was expressed at significantly lower levels on activated CD8+ T cells than
on CD4 single positive T cells (Fig. 3 C). RT-PCR analysis revealed that CD4 mRNA was undetectable in freshly
isolated CD8+ T cells, but clearly induced after anti-CD3/
B7.1 stimulation, suggesting transcriptional activation of
the CD4 gene (Fig. 3 B). In experiments designed to determine the minimal requirements for CD4 expression on
neonatal CD8+ T cells, we found that: (a) TCR/CD3-mediated signals were sufficient to induce low levels of CD4,
but that CD4 induction was markedly enhanced by CD28
costimulation (Fig. 3 D), and (b) cellular proliferation was
neither sufficient or necessary. Indeed, CD4 was not expressed on proliferating CD8+ T cells stimulated with anti-CD2 together with anti-CD28 mAbs and IL-2, whereas it
was induced on irradiated (nonproliferating) anti-CD3/
B7.1-stimulated cells (data not shown). Finally and importantly, CD4 was coexpressed on neonatal T cells activated under more physiological conditions, i.e., by antigenic
stimulation with allogeneic dendritic cells (Fig. 3 D). Thus,
physiological activation of umbilical cord blood CD8+ T
cells is associated with the coexpression of low to moderate levels of CD4, most likely as a result of CD4 gene transcriptional activation. Moreover, effective costimulation is
required for optimal induction of CD4 coexpression, presumably explaining why this phenomenon went unnoticed
in earlier studies. Consistent with their susceptibility to infection with M-tropic HIV, activated neonatal T cells expressed CCR5 mRNA (Fig. 3 B). Interestingly, they also
expressed CXCR4/fusin protein and mRNA (Fig. 3 E). As already mentioned, it is suggested that their resistance to
T-tropic HIV infection results from a postentry block in
viral replication (17). The ability to coexpress CD4 is
not a unique feature of neonatal CD8+ T cells, and indeed
a variable proportion (10-40%, n = 5) of CD8+ T cells isolated from adult blood expressed low levels of CD4 after
anti-CD3/B7.1 activation (data not shown). Since neonatal CD8+ T cells are immunologically naive whereas their
adult counterparts contain both naive and memory cells, it
will be of interest to examine CD4 expression and susceptibility to HIV infection in naive and memory CD8+ T cell
subsets. Preliminary results suggest that indeed, naive but
not memory adult CD8+ T cells may be induced to coexpress CD4 and become infectable by HIV.
These observations have potentially important biological
and clinical implications. They challenge the paradigm that
the CD4 gene is irreversibly silenced in extrathymic mature
single positive CD8-
/
TCR-
/
T lymphocytes and
raise the question of the role of CD4 in the biology of
CD8+ T cells. A small fraction (2-3%) of peripheral TCR-
/
T cells are CD8 CD4 double positive (22). In the
large majority of the cases, these cells are CD4bright CD8dull
and express CD8 as a CD8-
/
homodimer (23). In three
cases however, the double positive T cells were shown to
display the same phenotype as activated neonatal CD8+ T
cells, i.e., CD8-
/
bright CD4dull, indicating that cells expressing this phenotype exist in vivo (24). Finally, infection
of CD8+ T cells by human herpesvirus 6 was previously
shown to induce CD4 expression and confer susceptibility
to HIV infection (25). From a clinical point of view, our
finding may explain the recent reports that CD8+ T cells of
infected patients can harbor HIV-1 (26, 27). It is tempting
to relate the HIV susceptibility of neonatal CD8+ T cells to
the rapid progression of the disease in a large proportion of
infected neonates. Since viral replication is mainly controlled by CD8+ T cells, it seems reasonable to assume that
the infection of these cells may compromise the immune
response to HIV. Productive infection of primary HIV-specific CD8+ T cells may lead to an early disruption of the
Ag repertoire of HIV-specific CD8+ T cells. Infected
CD8+ T cells may be deleted directly, after supporting
high rates of viral replication, or indirectly by cytotoxic
HIV-specific CD8+ T cells. Early restriction of the antigen
repertoire would facilitate the emergence of HIV variants
expressing functionally important Ags that were recognized
by the deleted T cells (28). Future studies aiming to
verify in vivo some of these hypotheses and to confirm the
differential susceptibility of naive versus memory CD8+ T
cells to HIV infection should further our understanding of the disease and have important implications for the development of HIV vaccines.
Address correspondence to G. Delespesse, Université de Montréal, Centre de Recherche Louis-Charles Simard, Laboratoire de Recherche en Allergie (M4211-K), Hôpital Notre-Dame, 1560 Sherbrooke St. East,
Montreal, Quebec H2L 4M1, Canada. Phone: 514-281-6000, ext. 5395; Fax: 514-896-4753; E-Mail: delespeg{at}ere.umontreal.ca
The authors thank Dr. M. Sarfati (University of Montreal, Montreal, Canada) for her most helpful experimental suggestions and Drs. G. Shearer and C. Lane (National Institutes of Health, Bethesda, MD) for critical review of the manuscript. The secretarial assistance of Norma Del Bosco is greatly appreciated.
This work was supported in part by a Medical Research Council grant to G. Delespesse. J.L. Riley is supported by Army contract No. 17-93-V-3004.
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