ISSN 0269-9370 © 1999 Lippincott Williams & Wilkins
HAART in HIV-infected patients: restoration of
antigen-specific CD4 T-cell responses in vitro is
correlated with CD4 memory T-cell reconstitution,
whereas improvement in delayed type
hypersensitivity is related to a decrease in viraemia
Thomas Wendland, Hansjakob Furrera, Pietro L. Vernazzab,
Karin Frutig, Anna Christena, Lukas Matterc, Raffaele Malinvernia
and Werner J. Pichler
Objective: To analyse prospectively the effect of highly active antiretroviral
treatment (HAART) on CD4 T-cell responses in vitro and in vivo in HIV-infected
Design: Prospective study with 49 protease inhibitor-naive adult patients. Data were
collected at baseline and after 3 and 6 months of HAART.
Methods: In vitro CD4 T-cell reactivity was analysed by stimulation of peripheral
blood mononuclear cells with several antigens. In vivo CD4 T-cell reactivity (delayed
type hypersensitivity) was assessed by Multitest Merieux. Both measurements were
correlated to CD4 (memory) T-cell count and HIV-1 viraemia.
Results: Restoration of specific CD4 T-cell proliferation was observed in most
patients. The in vitro T-cell response was restored more frequently against antigens
to which the immune system is constantly exposed (Candida albicans,
Mycobacterium tuberculosis, M. avium) as compared with a low-exposure antigen
(tetanus toxoid). Overall, delayed type hypersensitivity detection rate increased
under HAART. Multivariate analysis showed improvement of antigen-specific T-cell
proliferation to be significantly associated with an increase in memory CD4 T-cells,
whereas improvement of the delayed type hypersensitivity response was associated
with a decrease in plasma HIV-1 RNA.
Conclusions: HAART for 6 months restored antigen-specific CD4 T-cell response to
several antigens. In vitro immune reconstitution was closely correlated with an
increase in memory CD4 cells. Restoration of delayed type hypersensitivity was
associated with suppression of viraemia. It appears that in addition to expansion of
memory CD4 cells, suppression of viraemia following HAART may allow an improved
inflammatory reaction, thus providing even stronger immune reconstitution.
© 1999 Lippincott Williams & Wilkins
AIDS 1999, 13:1857–1862
Keywords: CD4, cellular immunity, delayed type hypersensitivity,
opportunistic infections, combination therapy
From the Institute of Immunology and Allergology, the aAIDS Unit, Inselspital Bern, the bAIDS Unit, Kantonsspital St. Gallen,
and the cInstitute of Medical Microbiology, Inselspital Bern, Switzerland.
Sponsorship: Supported by the Swiss National Foundation grant no.: 31-50482.97 (National AIDS-research program of the
BAG, to W.J.P.) and 3233-048902.96 to P.V.
Correspondence to Werner J. Pichler, Clinic for Rheumatology, Clinical Immunology/Allergology, University Hospital Bern,
Freiburgstrasse, CH-3010 Bern, Switzerland.
Received: 8 May 1999; revised: 27 July 1999; accepted: 28 August 1999.
AIDS 1999, Vol 13 No 14
Infection with HIV is characterized by a high rate of
HIV replication in CD4 T cells and progressive loss
and functional defects of CD4 T cells . CD4 T cells
of HIV-infected patients fail to respond to HIV pro-
teins shortly after infection, and loose their antigen-
specific responsiveness to opportunistic pathogens and
to recall antigens; this is shown by a loss of delayed
type hypersensitivity (DTH) response and of antigen-
specific T-cell proliferation [2,3]. The reduced func-
tional CD4 T cells and the consecutive breakdown of
cellular immunity contribute to the emergence of
opportunistic infections and may facilitate HIV-
replication . Highly active antiretroviral therapy
(HAART) results in strong and sustained inhibition of
HIV replication  and is associated with a rapid rise in
CD4 T cells in patients with advanced disease .
However, little is known about the functional integrity
of the reconstituted T-cells. Some data suggest that in
patients with advanced immunodeficiency, reconsti-
tuted CD4 T cells respond to opportunistic pathogens
. However, it is not clear which antigen-specific
CD4 T-cell responses improve or recover in these
patients in vitro and in vivo. We conducted a prospective
study to analyse CD4 T-cell responses by lymphocyte
proliferation assays in vitro and by DTH reactivity in
Material and methods
Patients and study design
HIV-1-positive, protease inhibitor-naive patients from
two participating centres (AIDS Unit, University
Hospital Bern, and AIDS Unit, Kantonspital St. Gallen,
Switzerland) were asked to participate in the study.
Patients were stratified into two groups according
to their baseline CD4 cell count (below or above
250 × 106/l). Treatment was started with a protease
inhibitor-containing HAART regimen. CD4 cell
counts, HIV RNA concentration and CD4 T-cell
responses were measured prospectively at baseline, and
after 3 and 6 months of HAART. Ten HIV-negative,
untreated volunteers were used as control group. The
study was approved by the local ethical review boards
and all subjects gave written informed consent.
Phenotyping of CD4 and CD8 lymphocytes was per-
formed on fresh EDTA-blood samples by 2 colour flow
cytometry as described previously . CD4 memory
T-cells were identified by anti-CD45RO monoclonal
antibody (Becton-Dickinson, Basel, Switzerland).
Absolute CD4 and CD8 cell counts were calculated on
the basis of absolute lymphocyte counts (Coulter
Counter FTKS, Coulter, Hialeah, Florida, USA).
Mycobacterium tuberculosis purified protein derivative was
obtained from the Statens Serum Institute
(Copenhagen, Denmark). M. avium purified protein
derivate was a gift of J. Stenderup (Statens Serum
Institute). Candida albicans antigen was provided by the
Institute of Medical Microbiology, University of Bern,
Switzerland. Toxoplasma gondii antigen (strain RH) was
provided by B. Gottstein, Institute of Parasitology,
University of Bern. Tetanus toxoid was supplied by J.
Cryz, Serum und Impfinstitut Bern, Switzerland. All
antigens were tested negative for endotoxin by limulus
lysate assay. Optimal antigen concentrations were
determined in proliferation assays with peripheral-
blood mononuclear cells (PBMC) of healthy HIV-
Lymphocyte proliferation assay
PBMC were isolated from 20 ml heparinized blood
samples by density gradient centrifugation using Ficoll-
Hypaque (Pharmacia, Uppsala, Sweden). PBMC were
frozen in foetal calf serum supplemented with 10%
dimethylsulfoxide and stored in liquid nitrogen until
use. For lymphocyte cultures, samples collected at all
time-points were thawed simultaneously. PBMC were
adjusted to 1 × 106/ml in RPMI 1640 supplemented
with 10% human AB-serum. PBMC (1 × 106/well)
were cultured in triplicate in a total volume of 200 µl
in 96-well round-bottom plates for 5 days in a standard
[3H]thymidine proliferation assay. Stimulation indices
were calculated as c.p.m. in culture with antigen
divided by c.p.m. in culture without antigen. Specific
proliferation was defined as a stimulation index > 3 and
[3H]thymidine incorporation > 1000 c.p.m.
Testing of DTH was performed with Multitest-Mérieux
(Pasteur Mérieux, Paris, France) according to the manu-
6 months of HAART. Assessment of reaction was
always performed by one responsible investigator in the
two participating centres. A DTH response was consid-
ered positive when an induration at the site of antigen
application, but not at the control site, was palpable.
Multitest-Meriéux contains seven antigens: C. albicans,
tuberculin, tetanus toxoid, Proteus mirabilis, diphtheria,
Trichophyton and Streptococcus. As only tetanus toxoid,
Proteus, Candida and tuberculin produced positive and
reproducible responses in HIV-negative individuals,
these DTH responses were included in the analysis. The
same batch was used throughout the whole study period.
before and after
Measurement of HIV-1 RNA
HIV-1 RNA concentration in blood was quantified in
EDTA plasma by HIV reverse transcription
(RT)–PCR (Amplicor HIV Monitor; Hoffmann-
LaRoche, Basel Switzerland), with a limit of detection
of approximately 200 copies/ml.
CD4 T-cell response after HAART Wendland et al.
Statistical analysis on changes of HIV-RNA and CD4
T cell counts were performed by Wilcoxon rank test
(comparison of patients at identical time points).
Multivariate analysis was performed with stata software
(version 5.0, Stata corporation, College station, Texas,
USA). Changes in DTH or lymphocyte proliferative
responses to the different antigens were tested with the
two-sided McNemar test. Differences were considered
significant at P ≤ 0.05.
Patient characteristics are shown in Table 1. Sixty-five
patients were included in the study, but only
49 patients concluded the 6 month treatment period
and were included in the final analysis. Thirty-three of
these 49 patients (67%) were treatment-naive, and
16 out of 49 were treatment-experienced but protease
inhibitor-naive. Twenty-four (49%) had a CD4 cell
count < 250 × 106/l.
Changes in viral load and CD4 T-cell counts
Table 2 shows changes in viral load and CD4 T-cell
counts. Reduction of viral load was not different
between patients. The median increase in total CD4
and in CD4 memory T-cell counts was higher in the
low than in the high CD4 stratum. This difference was
not statistically significant due to the high variability of
the individual responses.
CD4 T-cell proliferation
Patients in the low CD4 stratum
Antigen-specific proliferation was detectable in 12 out
of 24 (50%) patients in the low CD4 stratum before
HAART (Table 3). Recovery of antigen-specific CD4
T-cell proliferation was observed in most patients after
3 months of HAART (Fig. 1a). This response was
antigen-dependent and most pronounced with C.
albicans (0 versus 3 months, P = 0.003; 0 versus 6
months, P = 0.001), M. tuberculosis (0 versus 3 months,
P = 0,022; 0 versus 6 months, P = 0.012) and
M. avium (0 versus 3 months, P = 0,07; 0 versus 6
months, P = 0.039). A comparison of the improve-
ment between ubiquitous antigens and tetanus toxoid
revealed a significantly better response for Candida only
(3 months, P = 0.001; 6 months, P = 0.039). The
number of antigens to which PBMC of individual
patients responded increased from a median of 12.5%
before HAART to a median of 66% after 6 months of
therapy (P = 0.0001).
Patients in the high CD4 stratum
CD4 T-cell proliferation in response to opportunistic
antigens was constantly present in most patients before
initiation and during 6 months of HAART at a rate
comparable to that in HIV-negative controls (Fig. 1a).
Thus, a high proportion of patients with CD4 counts
Table 1. Patient characteristics.
CD4 T-cell count (× 106/l)
< 250> 250
Men who have sex with men
Injecting drug users
Centers for Disease
42 (31–63)35 (29–50)
Table 2. Changes in viral load and CD4 T-cells.
CD4 T-cell count (× 106/l)
P < 250
versus > 250< 250> 250
HIV-1 RNA [median (range)] (log10copies/ml plasma)
∆ 3 months
∆ 6 months
CD4 T cell count [median (range)] (× 106 cells/l)
∆ 3 months
∆ 6 months
CD4 memory T cell counta[median (range)] (× 106 cells/l)
∆ 3 months
∆ 6 months
aCD45RO T cells. NS, Not significant.
n = 24
n = 25
5.12 (2.00 to 5.93)
–2.32 (1.49 to –3.88)
–2.69 (1.49 to –3.88)
4.38 (2 to 5.22)
–2.16 (0 to –3.19)
–2.16 (0.52 to –3.19)
101 (10 to 249)
51 (–43 to 361)
125 (–46 to 433)
401 (255 to 800)
20 (–143 to 295)
49 (–145 to 515)
77 (8 to 172)
41 (–34 to 169)
73 (–41 to 135)
261 (92 to 464)
11 (–137 to 208)
–3 (–153 to 435)
AIDS 1999, Vol 13 No 14
> 250 × 106/l had functional CD4 T cells specific for
important pathogens despite ongoing viral replication.
DTH-testing was performed in 26 HIV-infected
patients before and after 6 months of HAART and in
parallel in eight HIV-negative individuals. The
response rate increased after 6 months of HAART but
was still inferior to the in vitro response rate and to the
DTH response in HIV-negative volunteers (Table 3).
Analysis of DTH responses to specific antigens (Fig. 1b)
revealed an increase with antigens in patients in the
high CD4 stratum. In contrast, in patients with low
CD4 counts, recovery of DTH reactivity was limited
to a frequently exposed antigen (Proteus). There was an
increased response to purified protein derivative of
(M. tuberculosis) in healthy individuals after 6 months,
probably due to a booster effect of the previous skin
testing in this BCG vaccinated population.
Predictors of improvement of antigen-specific
T-cell proliferation and DTH response
The factors associated with an improvement of prolif-
eration and DTH response were analysed in a logistic
model, which included the increase of total CD4
counts as well as memory CD4 cell count as binary
response variable, and the decrease in log10plasma
HIV-1 RNA as continuous variable between time 0
and after 6 months of treatment. Only patients with all
of the variables available were included in the analysis.
Fig. 2 shows the median changes in CD4, CD4 mem-
ory T-cell numbers and of HIV-1 RNA levels of
patients with and without improvement of proliferation
or DTH. Table 4 shows the multivariate analysis of
these data: improvement in antigen-specific T-cell pro-
liferation was associated significantly with an increase in
memory CD4 T-cell number but not with increase in
CD4 T-cell number or decrease in HIV-1 RNA. In
contrast, improvement in DTH response was associated
significantly only with a decrease in plasma HIV-1
Table 3. Improvement of CD4 T-cell reactivity in vitro and in vivo.
Antigen-specific T cell proliferation
CD4 cells < 250 × 106/l
CD4 cells > 250 × 106/l
CD4 cells < 250 × 106/l (n=17)
CD4 cells > 250 × 106/l (n=9)
The percentages of patients with specific T-cell proliferation to any
antigen, and the percentage of patients with delayed type hyper-
sensitivity (DTH) responses to Tetanus toxoid, purified protein
derivative of Mycobacterium tuberculosis, Candida albicans or
Proteus mirabilis are shown.
50 83 96
Fig. 1. CD4 T-cell response in vitro and in vivo. (a) Recovery of specific CD4 T-cell proliferation. (b) Recovery of DTH-
responses. The ordinate shows the percentage of HIV-infected patients and HIV-negative individuals with responses to the
HIV-patients with less
than 250 CD4 × 106/l
HIV-patients with more
than 250 CD4 × 106/l
HIV-negative, no HAART
CD4 T-cell response after HAART Wendland et al.
In this study HAART over a period of 6 months
improves specific CD4 T-cell responses in vitro and in
vivo in adult HIV-infected patients. Improvement of
antigen-specific CD4 T-cell proliferation was observed
for all opportunistic pathogens tested. Together with
previous observations [6,7], our data indicate that
HAART-induced improvement of CD4 T-cell
reactivity is directed against multiple, clinically relevant
pathogens. Restoration of antigen-specific T-cell pro-
liferation was more frequently observed than restora-
tion of DTH reactivity. Although intrinsic differences
among both tests for measuring cellular immune func-
tion may account for this discrepancy, the multivariate
analysis performed points to distinct biological mecha-
nisms occurring under HAART. In fact, improvement
of proliferation is easily explained by a higher number
of memory cells available which is not strictly associ-
ated with suppression of viraemia. On the other hand,
suppression of viraemia appears to be a prerequisite for
improvement of DTH. Several explanations are possi-
ble for the latter observation: High HIV-1 levels are
associated with elevated tumour necrosis factor-α
levels, which may disturb cell recruitment . HIV
itself may induce long-lasting changes in antigen-
presenting cell functions in the skin, thus altering
DTH. Furthermore, even if the CD4 cell reactivity is
reconstituted, high levels of HIV may still have a sup-
pressing effect on the rather complex cascade of events
following T-cell triggering which leads to a DTH
reaction in the skin. A possible mechanism for this
observation could be an interference by HIV virions or
soluble gp160 proteins with chemokine receptors
required for the recruitment of inflammatory cells .
However, our data need confirmation in a larger
sample of HIV-infected subjects under HAART.
CD4 T-cell responses in vitro were observed more fre-
quently after exposure to antigens of opportunistic
pathogens (in particular C. albicans) than with tetanus
toxoid before and after HAART. These findings sug-
gest that reactivity of circulating CD4 T-cells is skewed
in favour of highly exposed antigens in chronic HIV-
infection, in agreement with earlier observations
The differences seen in both patient strata regarding
CD4 T-cell proliferation to opportunistic pathogens
before initiation of HAART are in good correlation
with clinical data. For example, only 35% of patients
with CD4 cell counts < 250 × 106/l (a group at high
risk of severe Candida infections) but 96% of subjects
Table 4. Improvement of proliferation and delayed type hypersensitivity response before and after 6 months of HAART as predicted in a
logistic model including increase of absolute CD4 cell count and absolute memory CD4 cell count as binary response variable and decrease
in log10plasma HIV RNA/ml as continuous variable. Results of univariate and multivariate analysis including all listed variables are shown.
Improvement in proliferation
n = 46
Improvement in delayed type hypersensitivity
response n = 23
Univariate Multivariate UnivariateMultivariate
Variable Odds ratio
Increase in CD4 T cell count
Increase in CD4 T cell memory
count [median (range)]
Decrease in HIV-1 RNA
(per 1 log10decrease)
1.5 (0.3–6.8)0.6 0.3 (< 0.1–2.8) 0.3 1.0 (0.1–18)1.00.5 (< 0.1–956) 0.9
3.9 (1.1–14.0)0.047.0 (1.8–41.5) 0.031.1 (0.1–9.6)0.90.3 (< 0.1–21.5) 0.6
1.1 (0.7–1.7)0.6 0.6 (0.7–1.6) 0.9 2.8 (1.1–7.3)0.03 3.6 (1.2–10.2) 0.02
Fig. 2. Predictors of improvement of proliferation and
delayed type hypersensitivity (DTH) response. Box plot
representation of the median changes (± 25 to 75 per-
centiles; error bars indicate 10th and 90th percentiles) of
CD4, CD4 memory and HIV-1 RNA levels of patients after
6 months of HAART with and without improvement of pro-
liferation or DTH response.
[CD4 T-cells × 106/l]
[CD4 T-cells × 106/l]
AIDS 1999, Vol 13 No 14
with higher CD4 cell counts showed proliferation
against C. albicans. The rapid recovery of the Candida-
specific CD4 T-cell proliferation is in accordance with
epidemiological data showing a rapid decrease in the
incidence of candidal oesophagitis after a few months
of HAART treatment in patients with advanced
immune dysfunction . Furthermore, the beneficial
effect of HAART extends to other antigens as well, as
shown most recently after stopping primary and sec-
ondary prophylaxis against Pneumocystis carinii, and, pos-
sibly, T. gondii and cytomegalovirus [15–17].
In conclusion, HAART results in a significant restora-
tion of multi-specific CD4 T-cell responses, but the
degree of this effect is different when in vitro or in vivo
methods are used. Functional studies of CD4 T cells
might help to guide decisions regarding discontinuation
of prophylactic medication against opportunistic infec-
tions in patients with undetectable viral load under
The authors thank all patients for their participation
and the staff of the AIDS-units in Bern and St. Gallen
for their efforts making the study possible. Furthermore
we thank D. Züllig for excellent technical support and
M. Zanni, C. Burkhart and S. von Greyerz for helpful
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