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*
Cellular Immunology Unit, Centre for Biology and Molecular Pathology, Faculty of Medicine of Lisbon, Lisbon, Portugal; and Departments of
Infectious Diseases and
Medicine 2, Faculty of Medicine of Lisbon/University Hospital of Santa Maria, Lisbon, Portugal
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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+ cells was not significantly
reduced after 1 yr of effective therapy. Moreover, a detrimental role
of IL-4 is suggested by the association between an enhanced proportion
of IL-4-producing cells within the CD4 and particularly the CD8 subset
and viral load rebound. Finally, the kinetics of changes of cell
subsets assessed for simultaneous production of different cytokines
supports the view that cell reconstitution during highly active
antiretroviral therapy is initially due to redistribution of terminally
differentiated cells, followed by peripheral expansion of less
differentiated ones and a late progressive increase of the proportion
of functionally defined naive/memory precursor lymphocytes. These data
bring new support for the role of cytokine imbalances in AIDS
pathogenesis and may be relevant for the definition of
immunointervention targets. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HIV infection is associated with significant dysregulation of the cytokine network that contributes to AIDS immunopathogenesis either as a result of cytokine modulation of viral replication or due to effects on the immune system, namely suppression of T cell ability to mount appropriate immune responses, changes of cell susceptibility to apoptosis, and/or alterations of lymphocyte traffic 12, 13 . In spite of the importance of delineating the effects of HAART on HIV-associated cytokine imbalances, these have not to date been characterized.
Flow-cytometric single cell analysis of lymphokine production represents a powerful approach to investigate such effects since it permits simultaneous surface immunophenotyping and thus overcomes the effects of the altered representation of the different cell subsets on the cytokine profiles 14, 15, 16 . On the other hand, it circumvents the problem of cell selection during culture 14, 15, 16 , which allows the study of cell populations that exhibit low proliferation rates and are prone to apoptosis in vitro, such as the CD8+CD28- subset known to be markedly expanded in HIV-1 infection 17 . Furthermore, the sequential study during HAART of the relative proportion of the different cytokine-producing subsets may bring insights on the factors determining the homeostasis of these populations.
This new approach has contributed to a better definition of cytokine
imbalances in HIV-1 infection 18, 19, 20 . Our previous studies documented
major imbalances within the CD8 subset even in early stages of disease,
with low IL-2 and a marked increased frequency of IFN--producing
cells mainly with a CD28-negative phenotype. Unexpectedly, there were
limited alterations on the relative proportion of IL-2- and
IFN--producing cells within the CD4 subset in asymptomatic patients,
although a significant decrease in the potential to produce IL-4 was
documented in patients with more than 500/µl CD4 count 20 .
In this study, we investigated sequentially at the single cell level by flow cytometry, the effects of HAART on ex vivo lymphokine production profiles as a means to further delineate the role of cytokine imbalances on AIDS pathogenesis and to try to identify putative targets for immunointervention that may be required to achieve full immunologic reconstitution after HAART.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fourteen HIV-1-infected patients, six females and eight males, with a mean age of 32.6 ± 12.9 (range 20–61), were enrolled in the study. The mode of transmission was homosexual contact in five individuals, heterosexual contact in four, and i.v. drug addition in five patients who had stopped drug consumption at least 1 yr before the start of the study. Centers for Disease Control classification of HIV infection (1993) was as follows: eight patients in class A (2 A1, 5 A2, and 1 A3), five in class B (2 B1, 1 B2, and 2 B3), and one in class C1 (previously treated tuberculosis). At start of treatment, the mean CD4 counts were 378 ± 219/µl (range 11–828) with a documented decline in CD4 T cell counts during the previous year of the study (mean CD4 counts 503 ± 160/µl 1 yr before). The mean viral load at time zero was 4.8 RNA log copies/ml (range 4.3–5.8), and the treatment protocol was lamivudine (150 mg bid), stavudine (40 mg bid), and nelfinavir (750–1000 mg bid). Eleven patients were virgin of previous antiretroviral therapy, and the other three had only received reverse-transcriptase inhibitors (zidovudine alone or in association with didanosine or zalcitabine). Immunologic and virologic studies were done at weeks 0, 2, 4, 8, 16, 24, 32, 40, and 48 of treatment.
mAbs and reagents
The following anti-human cytokine mAbs were used:
anti-IL-2, clone MQ1-17H12, rat IgG2a (R-phycoerythrin-conjugated
(PE)); anti-IFN-, clone 45.B3, mouse IgG1 (FITC conjugated);
IL-4, clone 8D4-8, mouse IgG1 (purified and PE); mouse IgG1 (FITC and
PE), rat IgG1 (PE), and rat IgG2a (PE) isotype controls; all were
purchased from PharMingen (San Diego, CA). The final concentrations of
the mAbs were determined by titration in the study conditions.
Anti-human CD3 (TriColor conjugated (TC)), CD8 (FITC, PE, and TC),
CD45RO (TC), CD69 (FITC and PE), and mouse IgG2a isotype control (FITC,
PE, and TC) mAbs were obtained from Caltag (South San Francisco, CA).
Anti-human CD4 (FITC), CD28 (PE), CD45RO (PE), and mouse IgG1 isotype
control (FITC and PE) mAbs were purchased from Becton Dickinson
(Mountain View, CA). PMA (Sigma, St. Louis, MO) and ionomycin (I;
Calbiochem, La Jolla, CA) were used at 50 and 500 ng/ml final
concentrations, respectively, and brefeldin A (BFA; Sigma) was used at
10 µg/ml.
Immunofluorescent staining
Immunofluorescent staining was performed as previously described
20 . Briefly, PBMCs were isolated from fresh heparinized blood by
Ficoll-Hypaque (Life Technologies, Gaithersburg, MD) gradient
centrifugation, immediately after venopuncture, and resuspended at
1 x 106 cells/ml in RPMI 1640 (Life Technologies),
10% pooled AB human serum (Sigma), 100 U/ml penicillin/100 µg/ml
streptomycin (Life Technologies), and 2 mM glutamine (Life
Technologies). PBMCs were cultured with PMA + I in the presence of BFA
for 4 h. After a wash in PBS (Sigma), cells were fixed with 2%
formaldehyde (Sigma) by incubation for 20 min at room temperature,
washed again in PBS, resuspended in PBS containing 1% BSA (Sigma) and
0.1% sodium azide (AZ; Sigma) (PBS/BSA/AZ), and stained
immediately for cytokines or stored at 4°C for up to 2 days. After
surface staining with anti-CD8, anti-CD28, anti-CD69,
anti-CD3, and anti-CD45RO mAbs, cells were washed with
PBS/BSA/AZ and permeabilized with PBS/1% BSA/0.5% saponin.
Intracellular staining was done with anti-IL-2, anti-IL-4,
anti-IFN-, and anti-CD69 mAbs by 30-min incubation at
room temperature. After two washes in saponin buffer, cells were washed
and resuspended in PBS/BSA/AZ.
Flow-cytometric analysis of intracellular cytokine
Ten thousand or fifty thousand events (samples stained for IL-4) were acquired in a FACSCalibur flow cytometer (Becton Dickinson), and five-parameter analysis was performed using Cellquest software. Analysis was done within a manual setting lymphogate, and thresholds were set according to isotype-matched negative controls, staining of unstimulated cells, and previous block of staining with unconjugated mAbs. Results were expressed as the percentage of cells of a particular T cell subset that stained positive for a given cytokine. Because of the previously documented rapid down-modulation of CD4 in response to phorbol esters 15 , the CD4 subset was identified as the CD8-CD3+ cells. The presence of BFA in the culture protected the cell surface from activation-induced changes in CD45RO expression, allowing analysis within the ex vivo CD45RO+ subset 15 . To exclude that differences in T cell activation could bias the cytokine profiles, CD69 was used to assess the level of PMA + I-induced activation of the cells and shown to be expressed in more than 95% of the T cells in all of the experiments. Lymphocyte surface phenotyping studies of noncultured PBMCs were done in parallel using the following mAb combinations: FITC-CD4/PE-CD8/TC-CD3, FITC-CD4/PE-CD28/TC-CD3, and FITC-CD4/PE-CD45RO/TC-CD3. Absolute numbers of lymphocyte subsets were found by multiplying their representation by the absolute lymphocyte count obtained at the clinical laboratory, and results are presented for the 14 patients at all of the different times of the study, except at week 2, in which absolute counts refer to nine patients.
Plasma viral RNA assay
Plasma viral RNA load was measured by reverse-transcriptase PCR (Ultrasensitive Test; Roche Molecular Systems, Brandchburg, NJ) and the threshold was 50 HIV-1 RNA copies/ml (1.7 log copies/ml).
Statistical analysis
The data are presented as arithmetic mean ± SEM. Baseline and follow-up data were compared using paired t test or Wilcoxon-matched pairs test, according to the type of distribution of the variables, and for data of different groups unpaired t test and Mann-Whitney test were used; p values <0.05 were considered significant. The Pearson’s correlation coefficient was used to determine the correlation between two variables. For correlation with viral load, the Spearman’s correlation coefficient was selected.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HAART induced a marked decrease in the plasma viral load, which
was kept below the limit of test detection (50 RNA copies/ml) after the
sixteenth week of therapy in nine patients and a parallel increase in
the absolute numbers of the peripheral blood CD4 and CD8 T cells in all
of the 14 patients studied (Table I
).
Furthermore, a significant enhancement in the proportion of CD4 within
the T cells was documented, leading to a progressive increase in the
CD4/CD8 ratio that reached statistical significance at week 24 after
start of treatment (0.58 ± 0.09 at baseline to 0.73 ± 0.11,
p = 0.003). No alterations were found in the frequency
of double-negative T cells (CD4-CD8-) as well
as of the double-positive T cells (CD4+CD8+).
After a transient increase in the proportion of CD45RO+
cells at the second week of therapy, a progressive decline in the
percentage of these cells was observed in the CD4 and CD8 subsets.
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A representative profile of the cytokine analysis is shown in Fig. 1.
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A). The analysis of absolute
counts revealed an initial marked increase in the number of both
IL-2-producing CD4 and CD8 T cells, although a sustained progressive
increase after 24 wk of treatment was only observed in the
IL-2-producing CD4 T cell counts (Fig. 2B). These
alterations paralleled the increase in CD4 and CD8 counts,
respectively. When the patients were divided according to the control
of viral replication into a group of nine with sustained viremia below
50 RNA copies/ml (cutoff of the test) and another with rebound viremia
after the initial drop induced by HAART, it was observed that the
increase in the proportion of IL-2-producing cells within the CD4
subset only reached significance in the group with sustained
suppression of viremia (Fig. 2C).
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-producing cells
There was a trend for an increase in the proportion of
IFN--producing CD4 T cells during therapy, although the comparison
with baseline mean value does not reach statistical significance.
However, a significant increase in the number of peripheral blood
CD4+IFN-+ T cells was documented since the
eighth week of treatment (73 ± 12/µl at the start of therapy to
104 ± 13/µl, p = 0.01).
The change in the frequency of IFN-+ cells within the
CD8 T cell subset (baseline value: 52.8 ± 4.8%) showed an
initial slight increase until the sixteenth week of treatment, followed
by a decrease that did not reach statistical significance (Fig. 3A), and similar results were
documented for the IFN-+CD8+CD3+
cell counts (baseline value: 477 ± 127 cells/µl), as shown in
Fig. 3B. To investigate whether these relatively
nonimpressive changes in IFN- production within the CD8 subset were
the result of opposite effects of HAART on the IFN--producing
CD28+ and CD28- populations, a further
characterization of the phenotype of the CD8 subset was done. At time
zero, there was a marked expansion of IFN--producing CD28-negative
cells within the CD8 subset (32.9 ± 10.4% in patients versus
9.5 ± 4.3% in healthy controls, p = 0.01), which
was only marginally reduced with treatment (no statistical
significance; Fig. 3C). On the other hand, HAART induced an
increase in the frequency of IFN--producing cells with a
CD28+ phenotype, statistically significant at weeks 8 and
16 (Fig. 3C). Moreover, a very significant decrease in the
percentage of non-IFN--producing CD28-negative cells was shown (Fig. 3E). Finally, a significant increase in the proportion of
CD28+ IFN-- cells, a profile found in the
majority of unprimed cells, was documented at week 24 of therapy and
thereafter (Fig. 3E). Analysis of the absolute cell numbers
revealed that the increase in CD8 counts was mostly due to cells
with a CD28+ phenotype, and that the number of circulating
CD28-negative IFN--producing cells did not diminish significantly
with HAART (Fig. 3, D, E, and F).
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At time zero, there was a negative correlation between the proportion of IL-4-producing CD4 cells and the CD4 counts (r = -0.8223, p = 0.0003) as well as with the percentage of peripheral blood CD4 T cells (r = -0.7499, p = 0.002). The frequency of IL-4-producing CD8 T cells also correlated negatively with the degree of CD4 depletion at baseline (r = -0.5545, p = 0.039 for CD4 counts, and r = -0.5975, p = 0.024 for the percentage of CD4 T cells). No significant correlation was observed with the viral load.
The analysis of the distribution of individual values of IL-4-producing
CD4 and CD8 T cells shows that 4 of 14 subjects have percentages
clearly above normal range for healthy controls (Fig. 4, A and B). Viral
load was not significantly different in these patients, although they
exhibited lower CD4 counts (121 ± 66 versus 480 ± 47
cells/µl, p = 0.001). Interestingly, in two of these
four patients, HAART induced a marked reduction in the percentage of
IL-4-producing CD4 cells, although without reaching normal levels, and
this was accompanied by a parallel decline in the hypereosinophilia
that these patients exhibited at baseline. The remaining 10 patients
had percentages of IL-4-producing CD4 cells falling within the normal
range, although their mean value was lower than that of healthy
subjects. Curiously, in these patients, HAART induced an increase in
the mean percentage of IL-4-producing T cells both within the CD4 and
the CD8 subsets that reached higher degree of significance when the
analysis was done within the memory/effector population
(CD45RO-positive cells).
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, C and D. When results were analyzed in terms of absolute counts of IL-4-producing CD4 and CD8 T cells, higher levels at baseline were found in patients with viral load rebound (13 ± 4.2 vs 8.8 ± 0.9 in the group with sustained control of viremia for IL-4+ CD4 counts, and 50 ± 21.7 vs 7.6 ± 1.6, p = 0.019, for IL-4+ CD8 T cell counts), and an increase of IL-4-producing CD4 and CD8 counts was observed in both subgroups during therapy.
Analysis of simultaneous production of IL-2 and IFN- within T
lymphocytes
Analysis of cells simultaneously stained for IL-2, IFN-, and
CD3 (Fig. 5A in terms of
relative proportions, and Fig. 5B in terms of absolute
numbers) revealed that there was an increase in the proportion of
IFN-+IL-2- T lymphocytes at the second week
of treatment, followed by a progressive decline. This suggests that the
early increase in T cell counts is predominantly due to terminally
differentiated cells (IFN-+IL-2-). The
proportion of cells with a IFN-+IL-2+
phenotype enhanced during therapy, and this enhancement reached
statistical significance at weeks 4–24. Thus, within the Th1 cell
population, therapy induced after the fourth week, a change from a
terminally differentiated phenotype
(IFN-+IL-2-) to a less differentiated one
(IFN-+IL-2+) that is known to have higher
proliferative responsiveness. Finally, the mean percentage of
IFN--IL-2+ cells, a phenotype largely
expressed by naive cells, showed a marked enhancement after week 24,
which reached significance at week 40. Analysis according to the degree
of viral load control showed that this increase in functionally defined
naive cells only reached statistical significance in the group of
patients with sustained suppression of viremia (15.34 ± 1.76% at
start of treatment to 19.73 ± 2.16% at week 40,
p = 0.005, in the group of patients with sustained
control of viral replication versus 14.69 ± 1.82% at start
of treatment to 14.69 ± 1.69% at week 40, in the group with
rebound viremia).
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+) within total T lymphocytes and
within the CD45RO+ subset
The simultaneous analysis of IL-4 and IFN- production
(Fig. 6, A and B)
revealed a significant enhancement at the fourth week of treatment of
the frequency of Th0 (IL-4+IFN-+) cells both
within the memory/effector subset (CD45RO+ gated cells) and
within total T lymphocytes. An increase of
IL-4+IFN-- cells was also documented,
although later than for the Th0 population. No significant differences
were found in the mean proportions of
IL-4-IFN-+ lymphocytes within both subsets
during the follow-up. Similar findings were documented when the
absolute counts of IL-4- and IFN--producing subsets were analyzed
(Fig. 7, A and B).
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-) cells was
significantly higher in the subgroup with viral load rebound. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After the first 4 wk of therapy, there was a progressive increase in
the proportion of IL-2-producing cells that was mostly due to a
significant enhancement in the frequency of cells with the potential to
simultaneously produce IL-2 and IFN-, a profile known to be
associated with a population of primed cells with higher proliferative
capacity 21, 22 . Parallel studies in the same cohort of patients
revealed that this is concomitant with the major recovery of in vitro
lymphocyte proliferative responses to different mitogenic stimuli,
namely anti-CD3 mAb, anti-CD3 plus anti-CD28 mAbs, PHA,
pokeweed mitogen, and PMA + I (data not shown). This sequence of
events would be consistent with a major role of peripheral cell
expansion in cell recovery, after the initial effect of HAART on
lymphocyte redistribution. Interestingly, this putative increase in
renewal T cell capacity is further supported by the recent
observation of an increase in the fraction of cells expressing the
marker of proliferation Ki67 in the peripheral blood and lymph nodes
during the same period after start of treatment 5 .
After week 24, the increasing frequency of IL-2-producing T cells was
mainly due to an enhancement of the proportion of cells producing IL-2,
but no IFN-, a profile largely described in unprimed lymphocytes and
possibly also present in memory precursor cells 15, 23, 24, 25 . Thus, the
increase in this population suggests an effective de novo generation of
lymphocytes, possibly by thymopoiesis recovery, and an increase in the
peripheral memory precursor pool, which may have important implications
to the functional recovery during therapy. The prethymic stem cell
differentiation is considered essential for the reconstitution of the
perturbed TCR repertoire in HIV infection 6, 7, 8, 9 , and thus, it is
important to note that only patients with sustained control of viremia
showed a significant recovery of the frequency of
IL-2+IFN-- T cells, which would be
consistent with the view that an effective suppression of viral
replication is important to achieve an immunologic reconstitution 6 .
Defective production of IL-2 has long been described in HIV-associated immunodeficiency 12, 13 and represented the rationale for the trials of IL-2 immunotherapy aimed at improving Th and T cytotoxic immune responses 26 . Our finding of an increase in the potential for IL-2 production after the fourth week of HAART represents a cellular basis for the functional T cell recovery, which until now had only been assessed in terms of in vitro lymphoproliferative responses 3, 8 .
Another important issue in HIV immunodeficiency has been the origin and
role of the major expansion of the CD8 subset observed during the
primary infection and persisting throughout the natural course of
disease 27, 28, 29, 30 . This expansion has been shown to be largely due to
atypical CD8 T cells considered to be an effector terminally
differentiated population on the basis of the increased cytolytic
activity 31 , the loss of the costimulatory molecule CD28 17 , the
lower proliferative capacity with reduced telomere length 32 as well
as on the cytokine production profile: IFN-+,
TNF-
+, IL-2- 20, 22 . Although the
generation of such cells is a subject of hot debate, most argue that
they are Ag driven and include a large number of HIV-specific CTLs
30, 31, 32, 33 . Somehow unexpectedly, our study showed that 48 wk of
sustained control of viral replication did not significantly reduce the
expansion of the IFN--producing CD8 T cells with a CD28-negative
phenotype. One possible explanation is that even minor degrees of viral
antigenic load are enough to maintain this expanded population.
However, this hypothesis is not supported by recent data using direct
staining of virus-specific CD8 cells with tetrameric complexes of MHC
and HIV peptides, which showed a reduction of HIV-specific CD8 cells
during HAART 33 . Alternatively, this population might include large
amounts of nonspecifically expanded CD8 T cells, which persistency, in
spite of HAART, could result from distinct cell survival requirements
and/or from a distinct array of adhesion molecules that leads to its
persistence in the intravascular compartment 11, 34 . In this respect,
it is worth noting that our data showed that the observed increase in
CD8 counts during HAART was largely due to CD28-positive cells.
In view of the role of IFN- in the regulation of cell recirculation
35 , some authors have suggested that the alterations in lymphocyte
traffic observed in HIV infection are due to the increase in IFN-
production 11, 36 . Our data showing a major increase in the absolute
counts of peripheral blood cell populations in the absence of a
reduction in the proportion of IFN--producing cells do not favor a
central role of IFN- in the traffic disturbances in HIV infection.
However, a note of caution is required, since this approach measures
the frequency of cells producing a given cytokine and, therefore, it is
not possible to exclude that during HAART the amount of effective
cytokine secretion per cell decreases, either as a result of the
reduction of HIV viral Ag stimuli or due to the lower level of
bystander lymphocyte activation.
The finding of an increased proportion of IL-4+ cells within the CD4 and, more significantly, within the CD8 subset of patients who did not achieve a sustained control of viral replication, favors a critical role of IL-4 in the disease outcome after HAART that warrants further investigation. Particularly interesting in this context are the recent data showing that IL-4 modulates the chemokine receptor expression on T cells 37, 38 , which suggests a role of IL-4 in the phenotypic switch from nonsyncytia-inducing/CCR5 using virus to syncytia-inducing/CXCR4 using virus that has been associated with disease progression and an adverse response to treatment 38 . Our findings also agree with previous observations by Maggi et al. suggesting a deleterious role of Th2-like CD8 T cells in HIV-1 infection 39 .
The effects of HAART on the potential for cytokine production analyzed
at single cell level by flow cytometry are not consistent with a simple
Th1 to Th2 shift model 12 . However, it is important to stress that a
recovery of Th1 cells, as assessed by the production of IL-2 and
IFN-, was documented, and moreover, that levels of IL-4 above the
normal range seem to be associated with viral rebound during HAART,
which represent observations fitting into the general cytokine shift
model.
In conclusion, the sequence of recovery of T cell subsets defined by cytokine profiles during HAART is consistent with an early cell redistribution, followed by peripheral cell expansion and a late de novo lymphocyte production that is associated with an effective suppression of viral replication. Moreover, these data bring new support for the role of cytokine imbalances in AIDS pathogenesis and are relevant for the definition of immunointervention targets.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Rui M. M. Victorino, Department of Medicine 2, Faculty of Medicine of Lisbon/University Hospital of Santa Maria, Av. Prof Egas Moniz 1600 Lisbon, Portugal. E-mail address:
3 Abbreviations used in this paper: HAART, highly active antiretroviral therapy; AZ, sodium azide; BFA, brefeldin A; I, ionomycin; PE, phycoerythrin; TC, TriColor conjugated.
Received for publication September 2, 1998. Accepted for publication December 16, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(IFN-) production as HIV-1 associated markers of immune dysfunction. Clin. Exp. Immunol. 111:257.[Medline]
. Annu. Rev. Immunol. 15:749.[Medline]
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) and CD8 (
) T cell subsets. B, Mean absolute
counts of IL-2-producing CD4 (
). Statistic significance of the follow-up values in
comparison with baseline values: *, p < 0.05;
**, p < 0.01; and ***,
p < 0.001.
) indicates mean values of healthy
controls. Statistic significance of the follow-up values in comparison
with baseline values: *, p < 0.05; **,
p < 0.01; and ***, p <
0.001. Statistic significance between the two groups of patients in
each time of study: §, p < 0.05; §§,
p < 0.01; and §§§, p <
0.001.
) within CD3+
gated lymphocytes. B, Results are expressed as the mean
absolute counts of IFN-
