![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Clinical and Diagnostic Laboratory Immunology, November 2001, p. 1225-1230, Vol. 8, No. 6
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.6.1225-1230.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Comparative Analysis of Human Cytomegalovirus-Specific
CD4+ T-Cell Frequency and Lymphoproliferative Response
in Human Immunodeficiency Virus-Positive Patients
Giampiero
Piccinini,1
Giuditta
Comolli,1,2
Emilia
Genini,1
Daniele
Lilleri,1
Roberto
Gulminetti,2
Rita
Maccario,3
Maria Grazia
Revello,1 and
Giuseppe
Gerna1,*
Servizio di
Virologia,1 Dipartimento di
Pediatria,3 and Laboratori Ricerca Area
Biotecnologie e Area Infettivologica,2 IRCCS
Policlinico San Matteo, Pavia, Italy
Received 23 May 2001/Returned for modification 26 July
2001/Accepted 14 August 2001
 |
ABSTRACT |
Evaluation of human cytomegalovirus (HCMV)-specific T-helper
immunity could contribute in optimizing anti-HCMV therapy in human
immunodeficiency virus (HIV)-infected patients. Testin the lymphoproliferative response (LPR) is the standard technique used to
evaluate T-helper response, but its use in the routine diagnostic laboratory setting can be problematic. The most promising new alternative technique is the determination of HCMV-specific
CD4+ T-cell frequency by flow cytometry detection of
intracellular cytokine production after short-term antigen-specific
activation of peripheral blood mononuclear cells. HCMV-specific LPR and
CD4+ T-cell frequency were compared in a group of 78 blood
samples from 65 HIV-infected patients. The results showed concordance in 80.7% of samples. In addition, comparative analysis of sequential blood samples from 13 HIV-infected patients showed that while in about
half of patients the T-helper HCMV-specific immune response remained
stable during highly active antiretroviral therapy (HAART), in
the other half declining levels of the HCMV-specific
CD4+-mediated immune response were determined by either one
or both assays. In conclusion, our data suggest that the determination of HCMV-specific CD4+ T-cell frequency can be considered a
valuable alternative to the LPR test for the detection of HCMV-specific
T-helper response in HIV-infected patients. It could facilitate wider
screening of anti-HCMV T-helper activity in HIV-infected patients, with potential benefits for clinicians in deciding strategies of
discontinuation or maintenance of anti-HCMV therapy.
| |
INTRODUCTION |
In the last few years, highly active
antiretroviral therapy (HAART) has greatly reduced the incidence of
human cytomegalovirus (HCMV) disease in human immunodeficiency virus
(HIV)-infected patients in Western countries (5, 19, 20).
However, at least two reasons suggest that careful monitoring of HCMV
disease in HIV-infected patients is still important: first, some cases of HCMV disease in patients with relatively high
CD4+ T-cell counts have been reported (7,
10, 11, 15); second, the guidelines for the discontinuation of
maintenance anti-HCMV therapy are mainly based on the
CD4+ T-cell count (12, 13, 16, 18, 24, 25,
27), regardless of any possible information on the
reconstitution of the HCMV-specific immune response. Evaluation of
specific anti-HCMV immunity could influence the clinical decision to
discontinue or maintain anti-HCMV therapy in patients with previous
HCMV disease and intermediate CD4+ T-cell counts.
In this respect, evaluation of the HCMV-specific T-helper response is
of particular interest (23). For several years, T-helper
immune response has been evaluated by testing the antigen-specific
lymphoproliferative response (LPR). However, the impact of the LPR
assay in guiding clinical decisions is still limited, since the assay
is time-consuming and poorly standardized and, being based on the use
of tritiated thymidine, requires specific containment measures and facilities.
The most promising new alternative technique for the determination of
HCMV-specific T-helper response is the evaluation of HCMV-specific
CD4+ T-cell frequency by flow cytometry detection
of intracellular cytokines after short-term antigen-specific activation
of peripheral blood mononuclear cells (PBMC), as recently reported
(1, 9, 14, 21, 26). This technique provides results in a
few hours, does not require the use of radioactive compounds, is easy
to standardize, and is applicable to frozen PBMC samples. Previous reports have shown that HCMV-specific CD4+ T
cells are almost totally polarized toward Th1 phenotype and therefore
that the frequency of CD4+ T cells producing
tumor necrosis factor alpha (TNF-
) or gamma interferon (IFN-
)
after exposure to HCMV antigens can be considered the overall frequency
of HCMV specific CD4+ T cells (21,
26). Some authors have described the analysis of HCMV-specific
CD4+ T-cell frequency in different stages of HIV
disease (14, 21), but LPR remains the most commonly used
test to evaluate the HCMV-specific T-helper response. At present, a
single report has been published comparing the HCMV-specific
CD4+ T-cell frequency and LPR in two groups
of HIV-seropositive patients (9).
In this study, we compared the two techniques by testing a series of
samples from HIV-infected patients. Samples from patients showing a
wide range of CD4+ T-cell counts, either with or
without HCMV-specific T-helper response, were examined. Our data
suggest that HCMV-specific CD4+ T-cell frequency
correlates with LPR and is a reliable alternative to the HCMV-specific
LPR test.
| |
MATERIALS AND METHODS |
Patients.
A total of 78 blood samples from 65 adult patients
(20 females, 45 males) that were both HIV and HCMV seropositive were
analyzed. The number of CD4+ T cells per
microliter of blood was assessed by the Ortho ImmunoCount Flow
Cytometer System (Raritan, N.J.), while the HIV type 1 (HIV-1) viral
load was determined by the bDNA technique (Bayer/Chiron Corp.,
Emeryville, Calif.). At first evaluation, of the 65 patients, 6 (3 with
and 3 without HCMV disease) were HAART naive with a median HIV load of
272,551 RNA copies/ml (range, <50 to 1,800,000 copies/ml) and 51 CD4+ T cells/µl (range, 1 to 221 cells), and 6 (3 with and 3 without HCMV disease) had been treated with short-term
HAART for 12 months (range, 8 to 20 months), reaching the level of <50
HIV RNA copies/ml and 271 CD4+ T cells/µl
(range, 65 to 410 cells/µl). In addition, of the remaining 53 patients treated with long-term HAART for 46 months (range, 29 to 57 months), 11 with HCMV disease were tested either during anti-HCMV
treatment (n = 6) or after its discontinuation
(n = 5), while 42 were examined in the absence of HCMV
disease. At initial observation, this patient group showed a median HIV
load of 59 RNA copies/ml (range, <50 to 150,070 copies/ml) and a
CD4+ T-cell count of 421/µl (range, 50 to 1,256 cells/µl).
PBMC preparation and storage.
PBMC were isolated by standard
Ficoll gradient centrifugation of heparinized blood samples and frozen
by resuspending 5 × 106 cell aliquots in
freezing medium (RPMI 1640 supplemented with 10% dimethyl sulfoxide
and 5% human albumin). When necessary, cells were thawed rapidly at
37°C and washed twice with RPMI 1640 containing 5% pooled human
serum (AB type; Euroclone, Wetherby, West York, United Kingdom).
Viability was evaluated by trypan blue exclusion staining. The rare
samples with a viability of <95% were discarded.
HCMV-specific lymphoproliferative assay.
Cells resuspended
in RPMI 1640 containing 5% pooled human serum (AB type) were plated in
triplicate at a 105 concentration (200 µl of
medium per well) in U-bottom 96-well microtiter plates. Viral and
control antigens were prepared by glycine buffer extraction (0.1 M, pH
9.6) of AD169 strain-infected and uninfected human embryonic lung
fibroblasts, followed by sonication and low-speed centrifugation.
Supernatants were stored in aliquots at 
80°C. Heat-inactivated HCMV
antigen (or control uninfected fibroblast antigen) was added at a
1:1,000 dilution (optimal stimulating concentration). After 6 days of
culture at 37°C in 5% CO2 atmosphere, 1 µCi
of [3H]thymidine per well was added, and the
cultures incubated for another 18 h. DNA-incorporated thymidine
was evaluated by harvesting cells and counting radioactivity in a
TopCount 100 system (Packard Instrument Co., Meriden, Conn.). The net
count-per-minute (cpm) response was calculated from the mean cpm of
wells with HCMV antigen minus the mean cpm of wells without HCMV
antigen. The stimulation index (SI) was the ratio of the mean cpm of
the wells with HCMV antigen to the mean cpm of the wells without HCMV antigen.
HCMV-specific CD4+ T-cell frequency evaluation.
HCMV-specific CD4+ T-cell frequency was
determined as previously described (21) with minor
modifications. Briefly, PBMC were resuspended in RPMI 1640 containing
10% fetal calf serum (Gibco, Grand Island, N.Y.) at 4 × 106 cells/ml. Half-milliliter aliquots were
dispensed in 15-ml polypropylene conical tubes (Becton Dickinson
Labware, Franklin Lane, N.J.). Then, 0.5 µg of anti-CD28 (Becton
Dickinson, San Jose, Calif.) and anti-CD49d (Southern Biotechnology
Associates, Birmingham, Ala.) monoclonal antibodies were added to each
aliquot. Next, 40 µl of a commercially available HCMV complement
fixation antigen preparation (AD169 strain grown in human embryonic
fibroblasts; BioWhittaker, Walkersville, Md.) at its optimal dilution
or its negative control was added as a specific stimulus; in some
experiments, staphylococcal enterotoxin B (SEB; Sigma, St. Louis, Mo.)
at 2 µg/ml was used as an alternative stimulus. Tubes were incubated 1 h at 37°C in a slant position and then brefeldin A (Sigma)
resuspended in ethanol was added at a 10-µg/ml final concentration.
Cells were incubated for an additional 14 h under the same
conditions, washed once with cold phosphate-buffered saline containing
2 mM EDTA, fixed and permeabilized by using a Fix & Perm kit (Caltag, Burlingame, Calif.) according to the manufacturer's instructions, and
then labeled with the following monoclonal antibodies (Becton Dickinson): anti-CD4-fluorescein isothiocyanate,
anti-TNF--phycoerythrin (PE), or anti-IFN--PE plus
anti-CD69-PerCP. Cell suspensions were analyzed with a FACSCalibur flow
cytometer (Becton Dickinson) equipped with CellQuest software. Viable
lymphocytes were identified by scattering parameters. For each sample,
50,000 viable CD4+ T lymphocytes were evaluated.
CD4+ cells expressing TNF- or IFN- and CD69
were considered activated cells. The HCMV-specific
CD4+ frequency was calculated by subtracting the
value of the control sample incubated with control antigen
(consistently 
0.05%). The value of HCMV specific
CD4+ T cells per ml of blood was calculated by
multiplying the HCMV-specific CD4+ T-cell
frequency by the number of CD4+ T cells per
milliliter of blood.
Statistical analysis.
The correlation between absolute
CD4+ T-cell counts and LPR to HCMV or
HCMV-specific CD4+ T-cell frequency as well as
that between LPR to HCMV and HCMV-specific CD4+
T-cell frequency was determined by calculating the coefficient of
regression R in a univariate regression analysis.
| |
RESULTS |
Comparative analysis of LPR and HCMV-specific CD4+
T-cell frequency in fresh and frozen PBMC samples.
HCMV-specific
CD4+ T-cell frequency was first evaluated in both
fresh and frozen PBMC samples from seven HCMV-seropositive subjects
(six HIV-seropositive and one HIV-seronegative). TNF- was used as an
activation marker because it gave a sharper peak than IFN- in flow
cytometry analysis. The HCMV-specific CD4+ T-cell
frequency found in frozen samples was 80.9% ± 67.7% of fresh
samples. Overall, all of the five fresh samples that were considered
positive for the presence of HCMV-specific CD4+ T
cells (according to criteria described below) also remained positive
when evaluated frozen. Individual data are reported in Table
1.
When HCMV-specific LPR was compared in fresh and frozen PBMC from five
HIV-positive subjects with positive LPR response in fresh samples, the
net cpm of incorporated thymidine in frozen samples as a percentage of
fresh samples was 36.4% ± 31.8% (see Table 1 for individual data).
Two of these samples (subjects 7 and 8), which were positive as fresh
samples, became negative when frozen. Therefore, analysis of frozen
samples was considered reliable for HCMV-specific
CD4+ T-cell frequency but not for LPR. Thus, all
subsequent tests were performed on frozen samples for HCMV-specific
CD4+ T-cell evaluation and on fresh samples for LPR.
Reproducibility of HCMV-specific CD4+ T-cell
analysis.
PBMC samples from three normal subjects were frozen, and
the frequency of HCMV-specific CD4+ T cells was
evaluated in three independent test runs. The coefficients of variation
were 31.9, 33.4, and 28.6% for the three subjects.
Definition of criteria for determination of cutoffs for
HCMV-specific CD4+ T-cell frequency and LPR.
Nine
healthy HCMV-seropositive subjects and six healthy HCMV-seronegative
subjects were evaluated for their HCMV-specific CD4+ T-cell frequency (Table
2). Based on these results and on the consideration that a flow cytometry determination of cell frequency of
<0.1% was considered unreliable, we established the following criteria to define the presence of HCMV-reactive
CD4+ T cells by flow cytometry analysis: the
simultaneous presence of an HCMV-specific CD4+
T-cell frequency of 
0.1% and 400/ml of blood. In parallel, the LPR
response was evaluated in 23 samples from 10 immunocompetent HCMV-seropositive subjects and in 8 samples from 4 immunocompetent HCMV-seronegative subjects (Table 3).
Patients were considered responders (R) for LPR to HCMV when the SI was
3 and the net cpm was 3,000 and defined as nonresponders (NR) when
either one or both of these conditions were not satisfied. All of the
nine HCMV-seropositive healthy subjects tested by both assays were found to be positive for both LPR to HCMV and a measurable frequency of
HCMV-specific CD4+ T cells.
Comparison of HCMV-specific CD4+ T-cell frequency and
LPR in HIV-infected patients
Seventy-eight PBMC
samples from 65 HCMV-seropositive HIV-infected patients (13 patients
were evaluated twice) were tested by both techniques. In summary,
according to the criteria reported above, 43 samples (55.1%) were
found to be positive and 20 samples (25.6%) were found to be negative
by both assays. In addition, seven samples (9.0%) were found to be
positive for HCMV-specific CD4+ T-cell presence but
negative for LPR, whereas eight samples (10.4%) were negative for
HCMV-specific CD4+ T cells but positive for LPR (Table
4). Therefore, 80.7% of assayed samples
gave concordant results by both techniques. As for discordant results,
the evaluation of HCMV-specific CD4+ T-cell frequency was
repeated for most samples, confirming previous results.
In addition, further aliquots of the same samples positive for LPR and
negative for HCMV-specific CD4+ T-cell presence
were tested. At first, impairment of cytokine production (possibly due
to the freezing procedure) was excluded by evaluating the intracellular
TNF- production in seven samples by stimulating PBMC with SEB. The
percentage of SEB-responsive CD4+ T cells was
6.56 ± 1.6 (mean ± the standard deviation), i.e., only
slightly lower than that found in samples from healthy subjects, thus
indicating that even in discordant samples the vast majority of
CD4+ T cells could be stimulated in vitro.
Furthermore, an abnormal cytokine pattern production was excluded in
three samples by evaluating the intracellular production of IFN-
instead of the production of TNF-. The analysis of the expression of
the two cytokines gave similar results. Finally, to exclude a varying
reactivity to antigens of different sources, the HCMV-specific
frequency of three samples was determined by using the same HCMV
antigen preparation used for LPR that confirmed results obtained
previously with the commercial antigen preparation. In addition, the
commercial HCMV antigen was found to induce LPR to a level comparable
to that seen with the antigen prepared in the laboratory.
However, when we examined discordant samples in detail, the few samples
with positive LPR but negative for HCMV-specific
CD4+ T-cell frequency appeared to show a
low-level LPR (net cpm < 10,000; see Fig.
1). In contrast, at least three samples
had an HCMV-specific CD4+ T-cell frequency of
1% but a totally impaired HCMV-specific LPR (Fig. 1).
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 1.
Correlation of HCMV-specific CD4+ T-cell
frequency and LPR as net cpm in 78 PBMC samples from 65 HIV-infected
patients. Dotted lines indicate the cutoffs for the HCMV-specific
CD4+ T-cell presence (0.1% of total CD4+ T
cells) and for HCMV-specific LPR (3,000 net cpm).
|
|
As for the different patient groups, concordant results were found in 6 of 6 treatment-naive patients (4 NR patients with CD4+ T cells at <50/µl and 2 R patients), 4 of
6 patients (3 NR, 1 R) treated with short-term HAART, 8 of 11 (7 R, 1 NR) patients with HCMV disease treated with long-term HAART, and 34 of
42 (28 R, 6 NR) patients treated with long-term HAART in the absence of
HCMV disease.
Correlation of HCMV-specific CD4+ T-cell count and
LPR.
The correlation of HCMV-specific CD4+
T-cell count and LPR was significant (R = 0.327, P = 0.003) (Fig. 1), whereas no significant correlation
was found between CD4+ T-cell count and either
the net cpm of LPR (Fig. 2A) or the
HCMV-specific CD4+ T-cell frequency (Fig. 2B). It
was evident that several patients with CD4+
T-cell count of <150/µl showed a high anti-HCMV T-helper activity, as documented by both assays. On the other hand, an even greater number
of patients with high CD4+ T-cell counts did not
show any anti-HCMV T-helper activity by either assay. These data
suggest that the CD4+ T-cell count alone is an
insufficient criterion for deciding upon strategies of anti-HCMV
therapy.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 2.
Correlation of CD4+ T-cell count per
microliter of whole blood and LPR (A) or HCMV-specific CD4+
T-cell frequency (B) in 78 PBMC samples from 65 HIV-infected patients.
Horizontal dotted lines indicate the cutoffs for the HCMV-LPR (3,000 net cpm, panel A) and for the HCMV-specific CD4+ T cells
(0.1% of total CD4+ T cells, panel B). In both panels, the
vertical dotted lines indicate the value of 150 CD4+ T
cells/µl of blood. Open symbols indicate samples with an SI of <3.0
(A) or HCMV-specific CD4+ T cells <400/ml of blood (B).
|
|
Sequential evaluation of HCMV-specific CD4+ T-cell
frequency and LPR in HIV-infected patients.
To investigate whether
the individual variability in the anti-HCMV T-helper response was due
to fluctuations of the immune response, 13 HIV-infected patients were
evaluated for both HCMV-specific CD4+ T-cell
frequency and LPR during a median period of 8 months (range, 2 to 10 months). At the time of the first evaluation, two of these patients
were HAART naive (patients 2 and 3), whereas the remaining patients had
been HAART treated for 3 years (except patient 1, who was treated
only for 9 months). Figure 3 shows
comparative data of LPR (panel A) and HCMV-specific
CD4+ T-cell frequency (panel B). Of the 13 patients, 2 (patients 3 and 4) did not respond by either assay over
time, and 5 (patients 2, 5, 6, 7, and 13) showed a substantially stable
response by both assays. On the other hand, 5 patients (patients 1, 8, 10, 11, and 12) showed declining levels of HCMV-specific T-helper response by both or either assay, and only one patient (patient 9)
displayed a positive response by flow cytometry at the time of the
second evaluation only (Fig. 3B).
View larger version (28K):
[in this window]
[in a new window]
|
FIG. 3.
HCMV-specific LPR (A) and CD4+ T-cell
frequency (B) in 13 HIV-infected patients tested during a period of 2 to 13 months (median, 8 months). All patients (except patients 1, 2, and 3) had been HAART treated for at least 3 years prior to first
evaluation. Only patients 3, 4, and 9 had a CD4+ T-cell
count of <150/µl and an HIV load of >10,000 HIV RNA copies/ml.  ,
first evaluation;  , second evaluation.
|
|
Clinically, patients 4 to 13 all had CD4+ T-cell
counts of <50/µl prior to HAART, and all had been treated with
long-term HAART at the time of the first examination. Of these,
patients 4 to 8 had had HCMV disease in the past and had been treated
with anti-HCMV therapy, which was discontinued for patients 7 and 8 prior to testing because of high CD4+ T-cell
counts, whereas patients 9 to 13 had no HCMV disease. All of these
patients had a negative LPR to HCMV prior to HAART.
| |
DISCUSSION |
HAART has been found to be effective in restoring
CD4+ T-cell counts and reconstituting the immune
system function in the majority of HIV patients (2, 5, 17, 19,
20, 22). However, some cases of HCMV retinitis in HIV patients
with high CD4+ T-cell counts have been reported
(7, 10, 15), suggesting that the HCMV-specific immune
response could not be restored in those patients, possibly due to
failure of reconstitution of the complete T-cell-receptor repertoire
(4, 6, 8). In addition, current guidelines suggest
maintenance or interruption of anti-HCMV therapy only based upon
CD4+ T-cell count (12, 13, 16, 18, 24, 25,
27). Furthermore, as shown in this study, some patients with low
CD4+ T-cell counts possess an apparently adequate
anti-HCMV immune response and so would not require anti-HCMV therapy.
As a matter of fact, one of the major aims of research in this field is
to develop an immunoassay that can evaluate the HCMV-specific immune
response (in particular, the T-helper response) and predict which
patients will relapse or reactivate HCMV disease. Lack of this
information is partially due to the poor standardization and the large
intrinsic variability of the most common method, the LPR assay. In this
respect, one of the most promising alternative techniques is the
evaluation of HCMV-specific CD4+ T-cell frequency
by cytokine flow cytometry. Despite the fact that this technique has
been already used in several clinical studies (1, 14, 21,
26), its correlation with the well-known LPR assay has, to our
knowledge, only preliminarily been reported (9).
The most interesting finding of the present study is that, in a
population of HIV-seropositive patients with a wide range of
CD4+ T-cell counts, analysis of presence of
HCMV-specific T-helper response by LPR and HCMV-specific
CD4+ T-cell frequency gave concordant results in
more than 80% of cases. Agreement between the two assays is only
slightly higher than that reported in a recent study, in which the
HCMV-specific CD4+ T-cell frequency was
determined by HCMV-specific stimulation of whole blood instead of PBMC
(9). The lack of an even higher concordance between the
two techniques could be explained by considering the two groups of
discordant results separately. In the group of patients positive for
LPR but negative for HCMV-specific CD4+ T-cell
frequency, the positivity for LPR was consistently weak. As a matter of
fact, it is possible that in a few samples very rare HCMV-specific
CD4+ T cells are present with a high
proliferative potential that can be detected by LPR but not by flow
cytometry analysis. On the other hand, in a few samples HCMV-specific
CD4+ T-cell presence was detected (in two cases,
at high frequency), but the same PBMC samples were not able to
proliferate in vitro in response to HCMV antigen. This discordance
could be explained by considering that LPR measures the expansion of
several antigen-specific T-cell clones, displaying different effector
functions, whereas the HCMV-specific CD4+ T-cell
frequency was calculated by taking into account only TNF-- and/or IFN--producing cells. It is conceivable that HIV-positive individuals showing a negative LPR, in the presence of a measurable frequency of HCMV-specific CD4+ T-cell frequency,
lack some other antigen-specific T-cell subsets, such as IL-2-producing
cells, that are necessary for optimal T-cell expansion. Alternative
explanations could include (i) a suboptimal antigen-presenting-cell
function, which might be inadequate for sustaining T-cell expansion but
sufficient to activate TNF-- and/or IFN--producing cells, and
(ii) the presence of a high proportion of HCMV-specific T cells with
suppressor function that can inhibit lymphocyte proliferation
(3).
The evidence that the HCMV-specific LPR assay and the evaluation of
HCMV-specific CD4+ T-cell frequency in a
proportion of HIV-positive individuals give rise to discrepant results
suggests that the evaluation of HCMV-specific immune recovery after
HAART therapy should include both assays at best. Nevertheless, the
results of the present study suggest that, whenever the availability of
PBMC is not sufficient to perform both assays, the evaluation of
HCMV-specific CD4+ T-cell frequency is as
reliable as LPR.
The concordance of results obtained by the two assays on the great
majority of samples tested was confirmed by the similarity of results
found in the short-term follow-up of the 13 HIV-infected patients
tested some months apart. While a fair proportion of patients showed
stable results, another substantial aliquot displayed results showing
declining levels of the HCMV-specific CD4+
T-cell-mediated immune response by either one or both assays after 3 to
4 years of HAART. These findings are in agreement with results recently
acquired in our laboratory showing a decrease or loss of the rescued
LPR to HCMV in AIDS patients after 3 to 4 years of HAART (unpublished data).
Present data do not provide an explanation for this phenomenon.
However, it is conceivable that immune recovery achieved after HAART
therapy might bring about a decrease in the frequency of HCMV
reactivation and, as a consequence, a reduction in the number of
circulating HCMV-specific T cells, which are no longer stimulated by
chronic antigen exposure.
The significant correlation of CD4+ T-cell
frequency and LPR supports the concept that the two assays may be
interchangeable. On the other hand, the lack of correlation between
CD4+ T-cell count and either the
CD4+ T-cell frequency or LPR documents that the
absolute CD4+ T-cell count and the other two
assays do not express a comparable immunological condition, thus
indicating that the CD4+ T-cell count alone is
insufficient for deciding upon strategies of discontinuation of
anti-HCMV therapy in AIDS patients with HCMV retinitis.
In conclusion, our data suggest that the determination of HCMV-specific
CD4+ T-cell frequency can be considered a valid
alternative to the LPR test for the detection of HCMV-specific T-helper
response in HIV-infected patients. This technique offers evident
advantages over LPR. It does not require the use of radioactive
compounds, and it is faster and easier to standardize. Furthermore, it
could facilitate wider screening of anti-HCMV T-helper activity in
HIV-infected patients, with potential benefits for clinicians in
deciding strategies of discontinuation or maintenance of anti-HCMV therapy.
| |
ACKNOWLEDGMENTS |
We thank Linda D'Arrigo for revision of the English. In
addition, we thank Maurizio Parea for helpful discussion of data. We
are indebted to Roberto Gentile for technical assistance.
This work was supported by Ministero della Sanità, Istituto
Superiore della Sanità, III progetto Nazionale AIDS (grant
50C.12), Ricerca Corrente IRCCS Policlinico San Matteo (grant 80425),
and Ricerca Finalizzata 820RFM99/01.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Servizio di
Virologia, IRCCS Policlinico San Matteo, 27100 Pavia, Italy. Phone:
39-0382-502644. Fax: 39-0382-502599. E-mail:
g.gerna{at}smatteo.pv.it.
| |
REFERENCES |
| 1.
|
Asanuma, H.,
M. Sharp,
H. T. Maecker,
V. C. Maino, and A. M. Arvin.
2000.
Frequencies of memory T cells specific for varicella-zoster virus, herpes simplex virus, and cytomegalovirus by intracellular detection of cytokine expression.
J. Infect. Dis.
181:859-866[CrossRef][Medline].
|
| 2.
|
Autran, B.,
G. Carcelaint,
T. S. Li,
G. Gorochov,
C. Blanc,
M. Renaud,
M. Durali,
D. Mathez,
V. Calvez,
J. Leibowitch,
C. Katlama, and P. Debre.
1999.
Restoration of the immune system with anti-retroviral therapy.
Immunol. Lett.
66:207-211[CrossRef][Medline].
|
| 3.
|
Boussiotis, V. A.,
E. Y. Tsai,
E. J. Yunis,
S. Thim,
J. C. Delgado,
C. C. Dascher,
A. Berezovskaya,
D. Rousset,
J. M. Reynes, and A. E. Goldfeld.
2000.
IL-10-producing T cells suppress immune responses in anergic tuberculosis patients.
J. Clin. Investig.
105:1317-1325[Medline].
|
| 4.
|
Connors, M.,
J. A. Kovacs,
S. Krevat,
J. C. Gea-Banacloche,
M. C. Sneller,
M. Flanigan,
J. A. Metcalf,
R. E. Walker,
J. Falloon,
M. Baseler,
R. Stevens,
I. Feuerstein,
H. Masur, and H. C. Lane.
1997.
HIV infection induces changes in CD4+ T-cell phenotype and depletions within the CD4+ T-cell repertoire that are not immediately restored by antiviral or immune-based therapies.
Nat. Med.
3:533-540[CrossRef][Medline].
|
| 5.
|
Deayton, J. R.,
P. Wilson,
C. A. Sabin,
C. C. Davey,
M. A. Johnson,
V. C. Emery, and P. D. Griffiths.
2000.
Changes in the natural history of cytomegalovirus retinitis following the introduction of highly active antiretroviral therapy.
AIDS
14:1163-1170[CrossRef][Medline].
|
| 6.
|
Gea-Banacloche, J. C.,
L. Martino,
J. M. Mican,
C. W. Hallahan,
M. Baseler,
R. Stevens,
L. Lambert,
M. Polis,
H. C. Lane, and M. Connors.
2000.
Longitudinal changes in CD4+ T cell antigen receptor diversity and naive/memory cell phenotype during 9 to 26 months of antiretroviral therapy of HIV-infected patients.
AIDS Res. Hum. Retrovir.
16:1877-1886[Medline].
|
| 7.
|
Gilquin, J.,
C. Piketty,
V. Thomas,
G. Gonzales-Canali,
L. Belec, and M. D. Kazatchkine.
1997.
Acute cytomegalovirus infection in AIDS patients with CD4 counts above 100 × 106 cells/l following combination antiretroviral therapy including protease inhibitors.
AIDS
13:1659-1660.
|
| 8.
|
Gorochov, G.,
A. U. Neumann,
A. Kereveur,
C. Parizot,
T. Li,
C. Katlama,
M. Karmochkine,
G. Raguin,
B. Autran, and P. Debre.
1998.
Perturbation of CD4+ and CD8+ T-cell repertoires during progression to AIDS and regulation of the CD4+ repertoire during antiviral therapy.
Nat. Med.
4:215-221[CrossRef][Medline].
|
| 9.
|
Jacobson, M. A.,
R. Schrier,
J. M. McCune,
F. J. Torriani,
G. N. Holland,
J. J. O'Donnell,
W. R. Freeman, and B. M. Bredt.
2001.
Cytomegalovirus (CMV)-specific CD4+ T lymphocyte immune function in long-term survivors of AIDS-related CMV end-organ disease who are receiving potent antiretroviral therapy.
J. Infect. Dis.
183:1399-1404[CrossRef][Medline].
|
| 10.
|
Jacobson, M. A.,
M. Zegans,
P. R. Pavan,
J. J. O'Donnell,
F. Sattler,
N. Rao,
S. Owens, and R. Pollard.
1997.
Cytomegalovirus retinitis after initiation of highly active antiretroviral therapy.
Lancet
349:1443-1445[CrossRef][Medline].
|
| 11.
|
Johnson, S. C.,
C. A. Benson,
D. W. Johnson, and A. Weinberg.
2001.
Recurrences of cytomegalovirus retinitis in a human immunodeficiency virus-infected patient, despite potent antiretroviral therapy and apparent immune reconstitution.
Clin. Infect. Dis.
32:815-819[CrossRef][Medline].
|
| 12.
|
Jouan, M.,
M. Saves,
R. Tubiana,
G. Carcelain,
N. Cassoux,
C. Aubron-Olivier,
A. M. Fillet,
M. Nciri,
B. Senechal,
G. Chene,
C. Tural,
S. Lasry,
B. Autran,
C. Katlama, and the RESTIMOP Study Team.
2001.
Discontinuation of maintenance therapy for cytomegalovirus retinitis in HIV-infected patients receiving highly active antiretroviral therapy.
AIDS
15:23-31[CrossRef][Medline].
|
| 13.
|
Kirk, O.,
J. D. Lundgren,
C. Pedersen,
H. Nielsen, and J. Gerstoft.
1999.
Can chemoprophylaxis against opportunistic infections be discontinued after an increase in CD4 cells induced by highly active antiretroviral therapy?
AIDS
13:1647-1651[CrossRef][Medline].
|
| 14.
|
Komanduri, K. V.,
M. N. Viswanathan,
E. D. Wieder,
D. K. Schmidt,
B. M. Bredt,
M. A. Jacobson, and J. M. McCune.
1998.
Restoration of cytomegalovirus-specific CD4+ T-lymphocyte responses after ganciclovir and highly active antiretroviral therapy in individuals infected with HIV-1.
Nat. Med.
4:953-956[CrossRef][Medline].
|
| 15.
|
Komanduri, K. V.,
J. Feinberg,
R. K. Hutchins,
R. D. Frame,
D. K. Schmidt,
M. N. Viswanathan,
J. P. Lalezari, and J. M. McCune.
2001.
Loss of cytomegalovirus-specific CD4+ T cell responses in human immunodeficiency virus type 1-infected patients with high CD4+ T cell counts and recurrent retinitis.
J. Infect. Dis.
183:1285-1289[CrossRef][Medline].
|
| 16.
|
Kovacs, J. A., and H. Masur.
2000.
Prophylaxis against opportunistic infections in patients with human immunodeficiency virus infection.
N. Engl. J. Med.
342:1416-1429[Free Full Text].
|
| 17.
|
Li, T. S.,
R. Tubiana,
C. Katlama,
V. Calvez,
H. Ait Mohand, and B. Autran.
1998.
Long-lasting recovery in CD4 T-cell function and viral-load reduction after highly active antiretroviral therapy in advanced HIV-1 disease.
Lancet
351:1682-1686[CrossRef][Medline].
|
| 18.
|
Macdonald, J. C.,
F. J. Torriani,
L. S. Morse,
M. P. Karavellas,
J. B. Reed, and W. R. Freeman.
1998.
Lack of reactivation of cytomegalovirus (CMV) retinitis after stopping CMV maintenance therapy in AIDS patients with sustained elevations in CD4 T cells in response to highly active antiretroviral therapy.
J. Infect. Dis.
177:1182-1187[Medline].
|
| 19.
|
Mocroft, A.,
C. Katlama,
A. M. Johnson,
C. Pradier,
F. Antunes,
F. Mulcahy,
A. Chiesi,
A. N. Phillips,
O. Kirk, and J. D. Lundgren.
2000.
AIDS across Europe, 1994-98: the EuroSIDA study.
Lancet
356:291-296[CrossRef][Medline].
|
| 20.
|
Palella, F. J., Jr.,
K. M. Delaney,
A. C. Moorman,
M. O. Loveless,
J. Fuhrer,
G. A. Satten,
D. J. Aschman, and S. D. Holmberg.
1998.
Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators.
N. Engl. J. Med.
338:853-860[Abstract/Free Full Text].
|
| 21.
|
Pitcher, C. J.,
C. Quittner,
D. M. Peterson,
M. Connors,
R. A. Koup,
V. C. Maino, and L. J. Picker.
1999.
HIV-1-specific CD4+ T cells are detectable in most individuals with active HIV-1 infection, but decline with prolonged viral suppression.
Nat. Med.
5:518-525[CrossRef][Medline].
|
| 22.
|
Pontesilli, O.,
S. Kerkhof-Garde,
N. G. Pakker,
D. W. Notermans,
M. T. Roos,
M. R. Klein,
S. A. Danner, and F. Miedema.
1999.
Antigen-specific T-lymphocyte proliferative responses during highly active antiretroviral therapy (HAART) of HIV-1 infection.
Immunol. Lett.
66:213-217[CrossRef][Medline].
|
| 23.
|
Schrier, R. D.,
W. R. Freeman,
C. A. Wiley, and J. A. McCutchan.
1995.
Immune predispositions for cytomegalovirus retinitis in AIDS. The HNRC Group.
J. Clin. Investig.
95:1741-1746.
|
| 24.
|
Tural, C.,
J. Romeu,
G. Sirera,
D. Andreu,
M. Conejero,
S. Ruiz,
A. Jou,
A. Bonjoch,
L. Ruiz,
A. Arno, and B. Clotet.
1998.
Long-lasting remission of cytomegalovirus retinitis without maintenance therapy in human immunodeficiency virus-infected patients.
J. Infect. Dis.
177:1080-1083[Medline].
|
| 25.
|
Vrabec, T. R.,
V. F. Baldassano, and S. M. Whitcup.
1998.
Discontinuation of maintenance therapy in patients with quiescent cytomegalovirus retinitis and elevated CD4+ counts.
Ophthalmology
105:1259-1264[CrossRef][Medline].
|
| 26.
|
Waldrop, S. L.,
C. J. Pitcher,
D. M. Peterson,
V. C. Maino, and L. J. Picker.
1997.
Determination of antigen-specific memory/effector CD4+ T cell frequencies by flow cytometry: evidence for a novel, antigen-specific homeostatic mechanism in HIV-associated immunodeficiency.
J. Clin. Investig.
99:1739-1750[Medline].
|
| 27.
|
Whitcup, S. M.,
E. Fortin,
A. S. Lindblad,
P. Griffiths,
J. A. Metcalf,
M. R. Robinson,
J. Manischewitz,
B. Baird,
C. Perry,
I. M. Kidd,
T. Vrabec,
R. T. Davey, Jr.,
J. Falloon,
R. E. Walker,
J. A. Kovacs,
H. C. Lane,
R. B. Nussenblatt,
J. Smith,
H. Masur, and M. A. Polis.
1999.
Discontinuation of anticytomegalovirus therapy in patients with HIV infection and cytomegalovirus retinitis.
JAMA
282:1633-1637[Abstract/Free Full Text].
|
Clinical and Diagnostic Laboratory Immunology, November 2001, p. 1225-1230, Vol. 8, No. 6
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.6.1225-1230.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Baldanti, F., Lilleri, D., Campanini, G., Comolli, G., Ridolfo, A. L., Rusconi, S., Gerna, G.
(2004). Human cytomegalovirus double resistance in a donor-positive/recipient-negative lung transplant patient with an impaired CD4-mediated specific immune response. J Antimicrob Chemother
53: 536-539
[Abstract]
[Full Text]
Top
Abstract
Introduction
