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Blood, Vol. 95 No. 5 (March 1), 2000:
pp. 1743-1751
IMMUNOBIOLOGY
Initiation of antiretroviral therapy during primary HIV-1
infection induces rapid stabilization of the T-cell receptor  chain repertoire and reduces the level of T-cell oligoclonality
Hugo Soudeyns,
Gabriele Campi,
G. Paolo Rizzardi,
Caterina Lenge,
James F. Demarest,
Giuseppe Tambussi,
Adriano Lazzarin,
Daniel Kaufmann,
Giulia Casorati,
Lawrence Corey, and
Giuseppe Pantaleo
From the Laboratory of AIDS Immunopathogenesis, Division of
Infectious Diseases, Department of Internal Medicine, Centre
Hospitalier Universitaire Vaudois, Lausanne, Switzerland; Unit of
Immunochemistry, DIBIT, and the Department of Infectious
Diseases, San Raffaele Scientific Institute, Milan,
Italy; Duke University Medical Center, Center for AIDS Research,
Durham, NC; Fred Hutchinson Cancer Research Center, Seattle, WA 98105.
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Abstract |
Major T-cell receptor  chain variable region (TCRBV) repertoire
perturbations are temporally associated with the down-regulation of
viremia during primary human immunodeficiency virus (HIV) infection and
with oligoclonal expansion and clonal exhaustion of HIV-specific cytotoxic T lymphocytes (CTLs). To determine whether initiation of
antiretroviral therapy (ART) or highly active antiretroviral therapy
(HAART) during primary infection influences the dynamics of
T-cell-mediated immune responses, the TCRBV repertoire was analyzed by
semiquantitative polymerase chain reaction in serial blood samples
obtained from 11 untreated and 11 ART-treated patients. Repertoire
variations were evaluated longitudinally. Stabilization of the TCRBV
repertoire was more consistently observed in treated as compared with
untreated patients. Furthermore, the extent and the rapidity of
stabilization were significantly different in treated versus untreated
patients. TCRBV repertoire stabilization was positively correlated with
the slope of HIV viremia in the treated group, suggesting an
association between repertoire stabilization and virologic response to
treatment. To test whether stabilization was associated with variations
in the clonal complexity of T-cell populations, T-cell receptor (TCR)
heteroduplex mobility shift assays (HMAs) were performed on sequential
samples from 4 HAART-treated subjects. Densitometric analysis of HMA
profiles showed a reduction in the number of TCR clonotypes in most
TCRBV families and a significant decrease in the total number of
clonotypes following 7 months of HAART. Furthermore, a biphasic decline
in HIV-specific but not heterologous CTL clones was observed. This
indicates that ART leads to a global reduction of CD8+
T-cell oligoclonality and significantly modulates the mobilization of
HIV-specific CTL during primary infection.
(Blood. 2000;95:1743-1751)
© 2000 by The American Society of Hematology.
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Introduction |
Acute or primary infection with human immunodeficiency
virus type 1 (HIV-1) is characterized by transient high-level viremia, which decreases significantly upon the emergence of the host
virus-specific immune response.1,2 Particularly effective
in the control of primary as well as chronic HIV-1 viremia is the
cellular immune response mediated by HIV-specific CD8+
cytotoxic T lymphocytes (CTLs).3-7 Important perturbations
in the T-cell receptor (TCR) chain variable region (TCRBV)
repertoire are frequently observed in subjects undergoing primary HIV
infection, consistent with the fact that HIV-specific CTL responses are
composed largely of variable numbers of oligoclonally expanded
CD8+ T-cell clones sharing common TCRBV
determinants.8,9 The relative frequency and magnitude of
these TCRBV repertoire perturbations have been correlated with the rate
of HIV disease progression, further reinforcing the notion that host
factors, including the qualitative nature of the CTL response,
represent important determinants of clinical outcome.10,11
CTL responses are unfortunately often tempered by viral escape
mechanisms, such as mutation of CTL epitopes and down-regulation of
cell-surface major histocompatibility complex class I molecules by
HIV-infected cells.12-16 In addition, study of the TCR
repertoire has revealed that a sizable proportion of HIV-specific CTL
clones expanded during primary infection9 was deleted in a
manner consistent with the occurrence of clonal exhaustion.17,18
In order to rapidly reduce circulating HIV levels, and to preserve host
virus-specific CD4+ helper T-cell responses, it has become
standard care to offer subjects with primary HIV infection the option
of immediately initiating treatment with potent antiretroviral
therapeutic combinations containing protease inhibitors, ie, highly
active antiretroviral therapy (HAART).19-21 Initiation of
HAART in chronic infection has been shown to induce rapid increases in
CD4+ T-cell counts, rapid improvements in immune function,
and a significant but incomplete normalization of the TCRBV repertoire
in both CD4+ and CD8+ T
cells.22-24 However, the effects of such a
regimen on the dynamics, diversity, and persistence of emerging
HIV-specific CTL responses have yet to be comprehensively studied. In
the present study, semiquantitative polymerase chain reaction (qPCR)
analysis was used to compare the perturbations in the TCRBV repertoire
seen in the absence and/or presence of antiretroviral therapy in 22 HIV-infected subjects with a diagnosis of primary infection, 11 in the
untreated and 11 in the treated group. The
relative complexity of the T-cell subset was compared prior
to and after initiation of HAART by means of TCR heteroduplex
mobility shift assays (HMAs) and imaging densitometry. Finally, the
relative representation of HIV-specific and heterologous CTL clones
was studied longitudinally following treatment of primary HIV infection
with HAART.
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Materials and methods |
We studied 23 subjects with primary HIV infection. All
subjects were enrolled within 12 weeks of initial presentation with symptoms consistent with primary HIV infection and acute retroviral syndrome.1,2 The criteria used for the diagnosis of primary HIV infection were (1) subjects who were HIV-viremic (antigen-capture assay, Abbott Laboratories, Abbott Park, IL) but remained
HIV-seronegative (Abbott enzyme-linked immunosorbent assay-negative,
Western blot-negative, or indeterminate) and (2) HIV-seropositive
individuals with a recent clinical history of primary HIV infection
(within 12 weeks from the onset of symptoms), detectable antigenemia,
and viremia greater than 50 000 HIV RNA copies per mL
plasma (Amplicor assay, Roche Diagnostics, Indianapolis, IN, or NASBA,
Organon-Teknika, Rome, Italy). In all cases, day 0 corresponds to the
time of presentation with symptoms of primary HIV infection. Peripheral
blood samples were obtained by venous puncture at regular intervals
over a mean follow-up period of 170 days (range = 63 to 414 days). In
12 subjects, treatment with different regimens of antiretroviral drugs
was readily initiated. These regimens consisted of (1)
zidovudine (AZT) and didanosine (ddI)
(n = 2); (2) AZT, 3TC, and indinavir (n = 7); and (3)
AZT, lamivudine (3TC), and saquinavir (n = 3) (Table
1). The untreated control group was
composed of 11 subjects, all of whom have been previously
described.8-10 Because of ethical concerns, study of
untreated control subjects was performed retrospectively, with the use
of serial cryopreserved samples collected prior to the advent of
current treatment recommendations for primary HIV infection. All
subjects were offered counseling and were required to provide full
informed consent, in accordance with guidelines from the medical review
board and/or ethics committee of all institutions concerned.
Peripheral blood mononuclear cells (PBMCs) were isolated from venous
blood samples by Ficoll-Hypaque density gradient centrifugation. Cell
samples were then frozen at 5 to 10 × 106 cells per
mL in 40% vol/vol fetal bovine serum (Gibco-Life
Technologies, Basel, Switzerland) and 10% vol/vol dimethyl sulfoxyde
(Sigma, St Louis, MO), prior to nucleic acid extraction. In some cases, cell pellets were solubilized before being frozen in 500 µL of 0.5% wt/vol N-lauroyl sarcosine, 25 mmol/L sodium citrate, pH 7, and 4 mol/L guanidine
thiocyanate (Sigma).
The TCRBV repertoire was analyzed in sequential unfractionated PBMC
samples collected at different time points after the onset of symptoms,
with the use of qPCR assay, as previously described.25,26 Briefly, RNA was extracted from 2 to 3 × 106 frozen
PBMCs with the use of Ultraspec RNA (Biotecx
Laboratories, Houston, TX) and were reverse transcribed in the presence
of p(dN)6 (20 µg per mL), 60 U avian
myeloblastosis virus reverse transcriptase (Pharmacia, Uppsala,
Sweden), 40 U RNasin (Promega, Madison, WI), and 1 mmol/L deoxynucleotides triphosphates.
TCRBV polymerase chain reaction (PCR) analysis used a panel of 5'
TCRBV-specific primers and a common 3' T cell receptor chain
constant region (TCRBC) primer. In each PCR reaction,
5' and 3' T cell receptor  chain constant region
(TCRAC) primers were included as internal amplification
controls. Sequences of TCRBV-specific and control oligonucleotides have
been reported elsewhere.25 To monitor the migration of PCR
products, 3' TCRBC and 3' TCRAC primers were end-labeled
with  -[32P] adenosine triphosphate (ATP)
(New England Nuclear, Boston, MA) with the use of T4
polynucleotide kinase (Boehringer-Mannheim, Rotkreuz, Switzerland). PCR
products were resolved on 10% polyacrylamide gels containing 7 mol/L urea (Sequagel, National Diagnostics, Atlanta, GA).
Radioactive signals were quantitated by means of an InstantImager
(Packard Instrument, Donners Grove, IL) and real-time volume integration.
CD8+ T cells were purified by flow cytometry with the use
of phycoerythrin-conjugated anti-CD8 monoclonal antibody (PharMingen, San Diego, CA) and were directly seeded (1 to 25 cells per well) into
96-well plates containing 50 000 irradiated allogeneic feeder cells,
as described.9 Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 20%
fetal bovine serum (Gibco-Life Technologies), 1 µg per
mL phytohemagglutinin (Sigma), and 40 U per
mL interleukin-2 (Boehringer-Mannheim). The CTL activity
of proliferating micropopulations was tested by standard
51Cr-release assay, with the use of autologous Epstein-Barr
virus-transformed B lymphoblastoid cell lines infected with
recombinant vaccinia viruses encoding specific HIV-1 proteins as
targets.8,9 Vaccinia isolates vvTG 3167 (HIV-1 Pol), vvTG
4113 (HIV-1 Rev), vvTG 3196 (HIV-1 Tat), vvTG 1147 (HIV-1 Nef), vvTG
1132 (HIV-1 gp120), vvTG 1144 (HIV-1 Gag), vvTG 9-1 (HIV-1 Env), and
vvTG 186P (Escherichia coli -gal negative control) were
kindly provided by Transgene SA (Strasbourg, France). The clonality of
HIV-specific and heterologous micropopulations was assessed by cloning
the TCR chains expressed by the CTL clones into pBluescript
KS+ (Stratagene Cloning Systems, La Jolla,
CA) and dideoxy-termination sequencing of the third
complementarity-determining region (CDR3), as described.9
CDR3 sequences were used to design clonotype-specific oligonucleotide
probes (see below).
Complementary DNA (cDNA) samples from sequential time
points were amplified with the use of a panel of TCRBV-specific
primers, as described.27,28 Amplification efficacy and
uniformity of yield were then assessed by agarose gel
electrophoresis. TCRBV-specific PCR products were mixed with 400 ng of
monomorphic extended carrier DNAs obtained by amplification of
corresponding TCRBV segments cloned into plasmid vectors, and were
denatured and reannealed slowly, as described.28 Products
of this reaction were then migrated on 8% nondenaturing polyacrylamide
gels (18 hours at 4° in a Bio-RAD Protean II Xi cell; Bio-RAD
Laboratories, Hercules, CA) and were blotted onto nylon
membranes (Hybond N+, Amersham-Pharmacia Biotech,
Dubendorf, Switzerland) by capillary transfer. Filters were hybridized
with -[32P]ATP end-labeled sense and
antisense oligonucleotides complementary to the extended part of the
carrier DNAs but not to the TCRBV-specific PCR products, in
6 × SSC, 1% bovine lacto-transfer technique optimizer (BLOTTO), 0.1% sodium dodecyl sulfate (SDS), and 5 mmol/L EDTA at 37°C.28 For heteroduplex
tracking of specific TCR chain clonotypes, oligonucleotide probes
complementary to the CDR3 region of HIV-specific and heterologous CTL
clones were used in an identical manner. Clonotype-specific probes used
were 3A11div (5'-ACC CTC AGG GGC GGT GTA-3'), 6G2div
(5'-TTG TGG TCG GGG GGG TCC TA-3'), 3B12div (5'-AGC
CAA ACA GAG GGG CTG TAC-3'), and 12.5B10div (5'-CCG TGG ACA
GGG GGC TCG TAT-3'). Filters were then washed twice in 6 × SSC, 0.1% SDS, for 30 minutes at 37°C and were exposed
to BioMax MR film (Kodak, Rochester, NY). Autoradiograms were scanned with a Bio-RAD Gel Doc 1000 imaging densitometer equipped with a
UV/white light conversion screen. Densitometric profile analysis was
performed with the Molecular Analyst software package (Bio-RAD Laboratories), with the use of a 3-mm-wide rectangular
averaging window and default automated peak detection settings (1% of
total area).
TCRBV repertoire data were transformed into point-to-point delta scores
( ), which represent the summation of absolute differences in the
relative expression levels of each individual TCRBV family between 2 consecutive time points.29 is therefore an index of
global repertoire variation between 2 time points, the value of being proportional to the extent of repertoire
variations.29 The slopes of over time were calculated
and were compared among different subjects with the use of the
Student t test, as the distribution of values did not
significantly deviate from the normal (P = .55,
Kolmogorov-Smirnov test). Correlations between the slope of over
time and the slope of viremia levels over time were determined by means
of linear regression analysis.
| |
Results |
TCRBV repertoire analysis in untreated subjects during primary
HIV infection
qPCR was used to analyze the TCRBV repertoire in serial samples of
total PBMCs from 11 subjects undergoing primary HIV
infection, who were studied prior to and after initiation of treatment
with various regimens of antiretroviral therapy (Table 1). This method can reproducibly quantitate differences in the expression levels of 24 TCRBV families.25,26 Longitudinal analysis of TCR chain repertoire perturbations was performed with the use of scores, which represent the sum of absolute differences between the
levels of expression of individual TCRBV families between consecutive time points. Therefore, reflects global repertoire variations, and
larger values are associated with larger repertoire variations, whereas low values indicate relatively small
variations.29 Thus, in a subject in whom several sequential
time points are tested, a negative slope of over time indicates
progressive repertoire stabilization, whereas a positive slope is
consistent with progressive repertoire destabilization.
As shown in Figure 1, values calculated
were frequently greater than 60, reflecting relatively large
point-to-point variations in the global levels of expression of the
various TCR chain familes.29 This is consistent with
the previously reported presence of multiple high-level perturbations
of the TCRBV repertoire in these patients.8-10 Of the
treated patients, 90.9% (10 of 11) showed negative slopes of over
time (mean slope =  0.130), indicating the occurence of
progressive repertoire stabilization in these subjects (Figure
1A). While negative slopes of over time were
seen in 63.6% (7 of 11) of the untreated patients, their slopes were significantly smaller than for the treated
patients (mean slope = 0.0124,
P = .036, Student t test) (Figure 1B). This indicates
that TCR repertoire stabilization occurred more rapidly in treated than
untreated patients (Figure
2A). Of note, the values of
slopes were positively correlated with the slopes of HIV-1
viremia in treated patients (n = 11, r = 0.525,
P < .05) but not in untreated subjects for whom viremia
data were available (n = 7, r = 0.174, P > .05)
(Table 1, Figure 2B and 2C). The mean follow-up times of patients in
the 2 groups were not significantly different (161.9 days in the
treated group compared with 188.9 days in the control arm;
P =.48, t test). As well, the mean number of
samples/time points tested per patient in the 2 groups did not
significantly differ (5.27 in the treated group compared with 5.18 in the control arm; P = .85, Student t test).
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| Fig 1.
Stabilization of the TCRBV repertoire following
initiation of antiretroviral therapy during primary HIV infection.
Levels of TCRBV expression determined by qPCR in 24 TCRBV families were
transformed into scores that represent the overall change in
TCRBV repertoire between 2 consecutive time points. The linear
regression line of over time was drawn, and the value of the slope
appears in the upper right corner. (A) Patients treated with
antiretroviral therapy or HAART during primary HIV-1 infection. (B)
Untreated primary infection patients.
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| Fig 2.
Stabilization of the TCRBV repertoire in patients treated
with antiretroviral therapy during primary HIV-1 infection.
The slope of the regression line of over time was calculated as in
Figure 1. HIV-1 viremia values were obtained by means of
quantitative-competitive PCR, NASBA (Organon-Teknika), or branched
DNA assay (Chiron, Emeryville CA), as described in
footnotes to Table 1. (A) TCRBV repertoire stabilization in untreated
subjects and patients treated with HAART during primary infection. The
mean slope in each subgroup is indicated by solid lines. (B)
Correlation between TCRBV repertoire stabilization and the slope of
HIV-1 viremia in untreated subjects during primary infection.
Significance of the correlation was tested by means of linear
regression analysis. (C) Correlation between TCRBV repertoire
stabilization and the slope of HIV-1 viremia in subjects treated with
antiretroviral therapy during primary infection. Significance of the
correlation was tested by means of linear regression analysis.
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Variations in T-cell oligoclonality during HAART treatment of
primary HIV infection
The fact that TCRBV-specific T-cell perturbations observed in
subjects with primary HIV infection result primarily from oligoclonal expansion and clonal deletion of CD8+ HIV-specific
CTL8,9 indicates that that the clonality of the initial
HIV-specific CTL response can affect the clinical outcome, possibly by
influencing the persistence of the CTL response.10 Therefore, in order to test whether HAART-associated TCRBV repertoire stabilization was associated with decreased oligoclonality (ie, increased complexity) of T-cell populations, TCR HMA was performed on
sequential samples from 4 primary infection patients (1166, 1168, 1155, and 1192), prior to and after initiation of HAART (zidovudine,
lamivudine, and indinavir). Since distinct TCRBV clonotypes segregate
as separate bands on HMA gels, this method allows simultaneous
determination of the levels of oligoclonality in all TCRBV families and
provides a direct assessment of the longitudinal changes in the clonal
composition of the T-cell populations. HMA is a practical alternative
to DNA sequencing-based diversity analysis, in which large numbers of
TCRBV molecular clones need to be examined in order to obtain a
comprehensive picture of the TCR repertoire
oligoclonality.8,9,30 HMA was performed on total PBMC
samples because it was shown that most if not all of the detectable
oligoclonality observed during primary HIV infection was limited to the
CD8+ subset,8,9 thereby limiting the usefulness
of T-cell subset-specific TCR HMA typing. Densitometric profile
analysis, combined with automated peak detection, was used to provide
an unbiased estimate of the number of different heteroduplexes in each
of the lanes of the HMA gels.
In patient 1166, profile analysis revealed the presence, in each of 26 TCRBV families and subfamilies tested, of between 4 to 12 different
TCRBV heterocomplexes, corresponding to discrete heteroduplex bands on
the HMA gels (Figure 3A and
3B). This value has been previously shown to be directly proportional
to the actual number of expanded TCRBV clonotypes present within a
clonally heterogenous TCRBV subset, and therefore represents an index
of the level of clonality of a complex T-cell population.28
After 7 months of HAART, changes in clonality profiles were readily detectable in all of the 4 patients in most of the TCRBV subsets (Figure 3A and 3B, data not shown). Densitometry and automated peak
detection revealed that patients 1166, 1168, 1155, and 1192 all
exhibited a reduced number of recognizable clonotypes per TCRBV family
in 19 of 26, 16 of 25, 16 of 24, and 14 of 26 TCRBV families tested,
respectively (Figure 3B and 3C, data not shown). As a consequence of
these reductions, there was a statistically significant decrease in
both patients in the total number of distinguishable clonotypes
following 7 months on HAART (230 before versus 181 after therapy in
patient 1166, P = .0012, Student t test; 201 before
versus 171 after therapy in patient 1168, P = .023; 194 before versus 163 after therapy in patient 1155, P = .015;
and 227 versus 192 after therapy in patient 1192, P = .015)
(Figure 3C). Although a global decrease in oligoclonality was observed, the number of detectable clonotypes was actually augmented in a few
TCRBV families in all subjects (TCRBV5, 12, 1352, and 22 in patient
1166; TCRBV8, 9, 11, 14 and 18 in patient 1168; TCRBV1, 8, 11 and 17 in
patient 1155; TCRBV7, 11, 1351, 16 and 21 in patient 1192), suggesting that the overall reduction in
oligoclonality was not incompatible with the concurrent emergence of
newly expanded T-cell clones (Figure 3C). Finally, the
appearance of a homogenously smeared background in the lanes of HMA
gels following 7 months of treatment further indicated that the TCRBV
repertoire was becoming progressively more polyclonal (Figure 3A and
3B). Taken together, these results are consistent with a notable
reduction in T-cell oligoclonality following 7 months of treatment with
HAART in these patients.
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| Fig 3.
Changes in the levels of T-cell oligoclonality following
induction of HAART during primary HIV infection.
The level of oligoclonality was determined for each TCRBV family by
using HMA, as described.28 (A) HMA gel hybridized with
Cb- and Cc-carrier-specific probes showing variations in
heteroduplex patterns for 26 TCRBV families and subfamilies in patient
1166, prior to and after initiation of HAART. (B) Computerized
densitometric analysis of the HMA autoradiogram shown in panel A. Triangles indicate discrete TCRBV heterocomplexes detected over the HMA
profiles with the use of automated peak discrimination as described in
"Materials and methods." TCRBV heterocomplex patterns prior to
(TCRBV A) and after (TCRBV B) therapy for each chain
V-region family from patient 1166 are shown. The actual
number of peaks detected appears in the bottom right corner.
(C) Summary of clonality analysis in
patients 1166, 1168, 1155, and 1192. Numbers of detectable clonotypes
are shown prior to and after the introduction of HAART, while the
decrease (closed boxes), stability (shaded boxes), or increase (open
boxes) in the levels of oligoclonality of respective TCRBV families is
indicated; nd indicates not done. Nomenclature of TCRBV families is
according to Arden et al.31
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Decay of HIV-specific CTL clones following HAART treatment of
primary HIV infection
To evaluate the effects of HAART on the persistence of HIV-specific
CTLs, HIV-specific CD8+ CTL populations were derived from
patient 0407 by limiting dilution. Populations 6G2, 3B12, and 12.5B10
displayed significant CTL activity, directed against autologous targets
infected with vaccinia virus recombinants expressing either HIV-1 gp120
or full-length HIV-1 Env (Figure 4A). In
contrast, population 3A11 did not display significant CTL activity
against targets expressing the HIV-1 Gag, Pol, Env, Tat, Rev, and Nef
gene products (Figure 4A). To assess the clonality of these
populations, the CDR3 region from the expressed TCR chain was
PCR-amplified and subcloned, and its DNA sequence was determined.
Unique functional TCR chains were detected in 3A11, 6G2, 3B12, and
12.5B10, indicating that these populations were distinct and largely
monoclonal (Figure 4B). Probes complementary to the CDR3 region of the
TCR chain expressed by these clonal populations were hybridized to
HMA gels, a method that allows the detection of 1:105 to
106 cells with specific clonotypes27,28 (Figure
5A and B). The relative representation of
each CTL clonal population was then determined by densitometric profile
analysis (Figure 5C). While the representation of non-HIV-specific
T-cell clone 3A11 increased during the study period, that of the 3 distinct HIV-Env-specific CTL clones sharply declined during 7 months
of HAART (Figure 5B and C). The homogeneous profiles obtained by using
carrier-specific probes indicate that these changes did not result from
quantitative differences in amplification efficiency or gel loading
(Figure 5A). The kinetics of decline were clearly biphasic, with an
initial t1/2 of 14.5, 8.82, and 13.0 days for clones 6G2,
3B12, and 12.5B10, respectively, during the first 2 weeks
(mean = 12.1 days). This was followed by a secondary decay phase with
a t1/2 of 273 and 313 days for clones 6G2 and 12.5B10,
respectively (mean = 293 days), while clone 3B12 did not exhibit a
secondary decay phase (positive slope) (Figure 5B and C). In contrast
to the rapid and total disappearance observed during clonal
exhaustion,9 all CTL clones remained detectable at the end
of the study period.
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| Fig 4.
Cytotoxic activity of HIV-specific and heterologous CTL
clones derived from patient 0407 during primary HIV infection.
(A) CTL activity of T-cell micropopulations derived by limiting
dilution was tested against autologous targets infected with a panel of
recombinant vaccinia viruses expressing HIV-1 proteins. The
significance threshold was set at 10% specific cytolytic activity. (B)
DNA sequence of the CDR3 (V[D]J) region of the TCR chain from the
4 CTL clones shown in A. The sequences used to design
clonotype-specific probes are boxed. Nomenclature of TCRBV families is
according to Arden et al.31
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| Fig 5.
Kinetics of representation of HIV-specific and
heterologous CTL clones in patient 0407 following treatment of primary
HIV infection with HAART.
Sequential cDNA samples from patient 0407 were amplified with the use
of cognate TCRBV-specific primers, and fractionated on HMA gels, with
cDNAs derived from CTL clones used as controls. These were probed with
(A) sense and antisense carrier-specific and (B) clonotype-specific
probes, as described in "Materials and methods." Homoduplex (H)
and heteroduplex (h) regions are shown. Heterocomplexes corresponding
to specific clonotypes are indicated by arrows. (C) Computerized
densitometric analysis of the HMA autoradiograms shown in panel B. The
y-axis corresponds to the area under the densitometric profile measured
over the heteroduplex region (h) shown in panel B.
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| |
Discussion |
It has been previously demonstrated that (1) oligoclonal expansions
of CD8+ T cells were commonly observed in HIV-infected
patients during primary infection8; (2) a large part of the
HIV-specific CTL activity measured during primary HIV infection was
mediated by cells within these expanded populations8,9; (3)
essentially all of the TCRBV repertoire variations seen in primary HIV
infection involved cells of the CD8+ subset8-10
(G.P., unpublished data, 1996); and (4) different patterns of TCR repertoire perturbationswere associated with distinct clinical outcomes, ie, slow versus rapid rates of CD4+
T-cell decline and different levels of viremia at a 1-year
endpoint.10 For these reasons, it was extremely important
to define whether initiation of antiretroviral therapy, and especially
HAART, during primary HIV infection influenced the patterns of TCRBV
repertoire perturbations usually associated with primary HIV-specific
CTL responses.
Our results have shown that a number of untreated primary infection
subjects exhibited TCRBV repertoire stabilization during primary
infection and after the transition to the chronic phase of the disease.
However, it is clear that primary infection patients treated with
antiretroviral therapy exhibited a more rapid rate of TCRBV repertoire
stabilization than untreated subjects, as indicated by a significantly
smaller slope of over time in these patients. Most interestingly,
values of the slope of were significantly correlated with the slope
of HIV-1 viremia in 11 patients treated with antiretroviral therapy,
suggesting the existence of a direct relationship between repertoire
stabilization and virologic response to treatment. It is conceivable
that TCRBV stabilization occurred as an indirect result of
antiretroviral therapy-induced reduction in viral replication and
circulating viral load, leading to a decline in the levels of
mobilization of CD8+ T cells and HIV-specific CTL in
particular. This is consistent with reported decreases in
CD8+ T-cell activation markers and with the progressive
decline in the frequency of HIV-1 peptide-specific CTL observed
following introduction of HAART during the course of chronic HIV
infection.22,32,33
A reduction in the number of HMA bands, as detected by imaging
densitometry, was also observed in 4 patients in whom primary HIV
infection was treated with HAART. As the large majority of clonotypes
detected with HMA in total PBMC represent clonal expansions of
CD8+ T cells,34-36 this reduction is consistent
with a partial but progressive transition from oligoclonality to
polyclonality of the CD8+ T-cell subset during the 7 months
spanning the 2 time points. This reduction was effected in a broad
range of TCRBV families, suggesting that it may involve not only
HIV-specific CTLs but also bystander-activated T cells. These data are
in accordance with the reduced oligoclonality of the CD8+
TCRBV repertoire previously reported in chronic HIV infection following
introduction of antiretroviral therapy.23,24 The appearance
of new discrete bands on heteroduplex gels may reflect emerging
CD8+ T-cell-mediated immune responses of undefined
antigeneic specificity. These new bands may also result from the
oligoclonal expansion of recently produced CD4+ T cells and
may therefore represent additional evidence of the resumption of
CD4+ T-cell production following introduction of
antiretroviral therapy.
Finally, when the representation of CTL clones was tested in a
longitudinal manner, a decline in the levels of HIV-specific CTL
clones, but not in the level of a non-HIV-specific T-cell clone, was
observed in response to suppression of HIV viremia. This decline was
biphasic, with a steep decrease in CTL representation in the first 2 weeks following introduction of HAART and a slower decline thereafter.
This may be related to different kinetics of disappearance of
HIV-specific CTL precursors and effectors, as previously
suggested.32,33 It is important to note that despite a
continuing decline, the 3 HIV-specific CTL clones analyzed were still
detectable at the end of the follow-up period, suggesting that viral
suppression may have prevented the occurrence of clonal exhaustion of
HIV-specific CTL.9
In conclusion, the results presented in this report indicate that
antiretroviral therapy significantly modulates the pattern of TCRBV
repertoire mobilization during primary infection and leads to a
substantial reduction in global CD8+ T-cell oligoclonality.
The rapid stabilization of the TCR repertoire observed in treated
subjects may reflect the transition of a larger proportion of CTL
effectors into memory cells and may delay or prevent clonal exhaustion
of the HIV-specific CTL clones that have become largely expanded during
primary infection.9 Furthermore, similar to what occurs in
chronic infection, the global reduction of CD8+ T-cell
oligoclonality induced by HAART during primary infection is associated
with a progressive reduction in the frequency ofcirculating HIV-specific CTL precursors and/or effectors. Therefore, it is conceivable that potentiation and long-term maintenance of a
diversified HIV-specific CTL repertoire during antiretroviral treatment
may require the development of therapeutic vaccine strategies to
achieve effective immune-mediated control of HIV
replication.37,38
| |
Acknowledgments |
The authors wish to thank D. Lee and L. Xie for their assistance with
data management, and P. Dellabona and A. S. Fauci for useful discussion
and critical reading of the manuscript.
| |
Footnotes |
Submitted July 6, 1999; accepted November 3, 1999.
Supported in part by the Santo-Suarez Foundation, the Swiss National
Science Foundation Grant 31-46032.95, and the National Institutes of
Health Grant NIH/1 UO1 AI41535-01.
H.S. is a Fellow of the Fonds de la recherche en santé du
Québec. G.P. is a scholar of the Giorgi-Cavaglieri Foundation.
Reprints: Giuseppe Pantaleo, Laboratory of AIDS
Immunopathogenesis, CHUV-Hôpital de Beaumont, 29 rue Beaumont,
1011 Lausanne, Switzerland; e-mail: giuseppe.pantaleo{at}chuv.hospvd.ch.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction