Download Low CD4+ T Cell Nadir Is an Independent Predictor of Lower HIV

BRIEF REPORT
Low CD4+ T Cell Nadir Is
an Independent Predictor of Lower
HIV-Specific Immune Responses
in Chronically HIV-1–Infected
Subjects Receiving Highly Active
Antiretroviral Therapy
M. Atif Siddique,1 Kelly E. Hartman,4 Ella Dragileva,4
Marla Dondero,5 Tebeb Gebretsadik,3 Ayumi Shintani,3
Laurence Peiperl,6 Fred Valentine,7 and Spyros A. Kalams1,2
1
Infectious Diseases Unit, Department of Medicine, and 2Department
of Microbiology and Immunology, Vanderbilt University Medical Center,
and 3Department of Biostatistics, Vanderbilt University, Nashville,
Tennessee; 4Partners AIDS Research Center, Massachusetts General
Hospital, Boston; 5AIDS Clinical Trial Group Operations Center, Silver
Spring, Maryland; 6University of California–San Francisco, San Francisco;
7
New York University Medical Center, New York, New York
The influence of CD4+ T cell nadirs on human immunodeficiency virus (HIV)–specific immune responses in subjects
with apparently normal CD4+ T cell counts is not known.
We evaluated the frequency of HIV-1–specific immune responses in a cohort of patients with complete viral suppression (HIV-1 RNA load, !50 copies/mL) who were receiving
highly active antiretroviral therapy and had a wide range of
CD4+ T cell nadirs. We found positive associations between
CD4+ T cell nadirs and the magnitude of HIV-specific CD8+
T cell responses (P p .02) and of T cell helper responses
(P p .04). These data show the CD4+ T cell nadir to be an
independent predictor of HIV-specific CD4+ and CD8+ T cell
responses in HIV-1–infected subjects with optimal suppression of viremia.
Highly active antiretroviral therapy (HAART) has significantly
improved the prognosis of HIV-1–infected individuals; however, recovery of CD4+ T cells is dependent on the timing of
initiation and on the duration of treatment. Although dramatic
increases in CD4+ T cell counts occur shortly after the initiation
of HAART [1–3], there is evidence that low CD4+ T cell nadirs
Received 8 December 2005; accepted 29 March 2006; electronically published 28 July
2006.
Reprints or correspondence: Dr. Spyros A. Kalams, 1161 21st Ave., MCN-A4103, Vanderbilt
University Medical Center, Nashville, TN 37232 ([email protected]).
The Journal of Infectious Diseases 2006; 194:661–5
2006 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2006/19405-0018$15.00
may be associated with immune defects that are difficult to
correct, and subjects with these defects may not recover CD4+
T cell counts to the point where prophylaxis for opportunistic
infections can be discontinued [4–6]. However, the relationship
between CD4+ T cell nadir and the magnitude of HIV-1–specific
cellular immune responses is not known. In the present study,
we evaluated the relationships among the CD4+ T cell nadir at
the time of initiation of potent antiretroviral therapy, the frequency of expression of activation markers (HLA-DR and CD38)
on CD4+ and CD8+ T cells, and the frequency of HIV-1–specific
CD8+ T cells that produce interferon (IFN)–g and lymphoproliferative responses.
Methods. A total of 84 HIV-1–infected individuals were
evaluated. These subjects were receiving stable treatment with
HAART and had had suppression of viremia to an HIV-1 RNA
load !50 copies/mL for at least 3 months. Viral loads were
determined using the Roche Amplicor Assay (version 1.5). HLA
class I typing was performed at the participating AIDS Clinical
Trial Group (ACTG) sites in tissue-typing laboratories using
polymerase chain reaction with sequence-specific primers. The
CD4+ T cell nadir was defined as the lowest recorded CD4+ T
cell value before the initiation of antiretroviral therapy. All individuals provided informed consent before participating in the
study. The cellular assays described below were not performed
on every subject, because of the limited availability of samples.
Lymphocyte proliferation assays were performed using fresh
peripheral-blood mononuclear cells (PBMCs) incubated with
p24 protein (1 mg/mL; Protein Sciences), IRC p24 antigen (Immune Response), p24 control protein (Protein Sciences), Candida antigen, phytohemagglutinin (PHA), or medium alone [7].
Results are expressed as D-counts per minute (Dcpm)—the
difference between counts in antigen-stimulated wells and control wells. There was a strong correlation between Dcpm and
the stimulation index (SI; r p 0.93; P ! .001; data not shown).
Only samples with Dcpm 1 1000 after PHA stimulation were
included in the analysis [7–9].
We synthesized 66 peptides (15–19 aa long, 10-aa overlap,
consensus sequence clade B 2001; Los Alamos HIV Database,
available at: http://hiv-web.lanl.gov) spanning HIV Gag protein
(MBS 396; Advanced Chemtech). In addition, we tested pepPotential conflicts of interest: none reported.
Financial support: National Institutes of Health (grants R01 AI39966 to S.A.K., 5U01AI027665
to F.V., program P30 AI 54999 to the Vanderbilt-Meharry Center for AIDS Research and P30
AI 027742 to the New York University Center for AIDS Research); OPO Foundation, Zurich,
Switzerland (support to M.A.S.); Elizabeth Glaser Pediatric AIDS Foundation (Elizabeth Glaser
Scientist Award to S.A.K.).
BRIEF REPORT • JID 2006:194 (1 September) • 661
tides corresponding to all HLA optimal HIV-1 cytotoxic T lymphocyte epitopes that were listed in the Los Alamos Database
(available at: http://www.hiv.lanl.gov/content/hiv-db/REVIEWS/
reviews.html) at the time of the study.
HIV-1–specific T cell responses were quantified using enzyme-linked immunospot (ELISpot) assay with frozen PBMCs
(0.7–1 ⫻ 10 5 cells/well) [10, 11]. For the evaluation of Gagspecific responses, pools of 5–6 overlapping Gag peptides were
placed in each well, and the total response to Gag was determined as the sum of positive pools after background subtraction. Class I peptides were tested individually at a final concentration of 20 mg/mL of each peptide [11]. Results are
expressed as the number of spot-forming cells per 106 PBMCs.
The number of specific IFN-g–secreting T cells was calculated
by subtracting the negative control value from the peptidespecific spot-forming cell count. Negative controls were always
!30 sfc/106 input cells. The positive controls were PBMCs incubated with PHA. Wells were considered to be positive if they
demonstrated at least 50 sfc/106 PBMCs after background subtraction and if the number of spot-forming cells was at least
3-fold higher than background. Only samples with a positive
response to PHA and a viability 170% were included in the
analysis. In addition, 3-color flow cytometry was performed in
accordance with standardized ACTG laboratory protocols with
anti–HLA-DR–fluorescein isothiocyanate, anti–CD38–phycoerythrin, and either anti–CD4– or anti–CD8–peridinin-chlorophyll-protein complex (all from Pharmingen).
Bivariate analyses were done using the Mann-Whitney U test,
and associations between continuous variables and HIV-1 immune-response variables were assessed using Spearman’s rank
correlation coefficient (rs). For the multivariate analysis, we
chose the sum of ELISpot responses to HLA optimal class I
peptide response and the magnitude of lymphoproliferative responses to HIV-1 p24 and IRC p24 antigens as dependent
outcomes. We did not have well-documented pretherapy viral
loads for the majority of subjects, so the impact of this variable
could not be tested. For each dependent variable, a multiple
linear-regression model was used that included the current CD4+
T cell value at the time of the immune assays or the CD4+ T
cell nadir, the number of days receiving antiretroviral therapy,
and CD4+ or CD8+ T cell activation status (HLA-DR and CD38
positive) for the estimation of the adjusted relationship with
HIV-1–specific immune responses.
Residual diagnostics were performed to ensure the accuracy
of the regression models, and dependent variables were transformed if necessary. For the total magnitude of responses against
HLA optimal class I peptide responses, a statistically significant
nonlinear relationship with CD4+ T cell nadir was found; therefore, a restricted cubic spline was used to fit a nonparametric
term in the model [12]. A linear b-coefficient was used for the
remainder of the variables. For all analyses, a 2-sided signifi662 • JID 2006:194 (1 September) • BRIEF REPORT
Figure 1. a, Relationship between current CD4+ T cell values and nadir.
CD4+ T cell values at the time of immune assays correlate with CD4+ T
cell nadirs in subjects only with CD4+ T cell nadirs 1200 cells/mm3. b,
Lower total magnitude of responses directed against HIV-1 peptides in
subjects with lower CD4+ T cell nadirs. On average, 20 individual peptides
were tested per subject, and samples from each subject recognized an
average of 4 peptides (range, 0–16 peptides/subject). In subjects with
CD4+ T cell nadirs 1200 cells/mm3, the median response was 661 sfc/
106 peripheral-blood mononuclear cells (PBMCs) (interquartile range [IQR],
338–2433 sfc/106 PBMCs), compared with 363 sfc/106 PBMCs (IQR, 50–
532 sfc/106 PBMCs) in subjects with low CD4+ T cell nadirs (P p .02).
c, Total magnitude of responses to pooled HIV-1 Gag peptides, plotted
as a function of CD4+ T cell nadir. The magnitude was lower in subjects
with CD4+T cell nadirs ⭐200 cells/mm3 (320 sfc/106 PBMCs (IQR, 144–
796 sfc/106 PBMCs) vs. 800 sfc/106 PBMCs (IQR, 458–1060 sfc/106
PBMCs) in subjects with CD4+T cell nadirs 1200 cells/mm3 (P p .10).
White triangles, CD4+T cell nadir ⭐200 cells/mm3; black triangles, CD4+T
cell nadir 1200 cells/mm3. CTL, cytotoxic T lymphocyte; IFN, interferon.
cance level of 5% was considered to indicate statistical significance. We used SAS software (version 8.02; SAS Institute) and
R software (version 2.0.0; available at: http://www.r-project.org),
and graphs were generated using SPSS software (version 13;
SPSS).
Results. We found CD4+ T cell nadirs to be highly associated with CD4+ T cell values at the time of the immune assays
(rs p 0.54; P ! .001) (figure 1a). However, despite this correlation in the group as a whole, there was no correlation between
current CD4+ T cell counts and nadirs in subjects with nadirs
⭐200 cells/mm3 (rs p ⫺0.04; P p .80), whereas the correlation
still held in the group with high CD4+ T cell nadirs (1200 cells/
mm3) (rs p 0.47; P p .001).
There was no association between the CD4+ T cell counts at
the time of the immune assays and the magnitude of IFN-g
ELISpot or lymphoproliferative responses (data not shown).
There was a nonlinear association between CD4+ T cell nadirs
and the magnitude of HIV-1 antigen–specific immune responses
with a threshold of ∼200 cells/mm3; therefore, we used this
cutoff value for subsequent analyses. This nadir threshold was
convenient because it split the groups relatively evenly and is
a clinically relevant CD4+ T cell value.
We evaluated 46 subjects for HIV-1–specific immune responses to peptides representing the optimal CD8+ T cell epitopes determined by each subject’s HLA class I A, B, and C
alleles. The sums of responses to optimal peptide epitopes in
subjects with high CD4+ T cell nadirs (1200 cells/mm3 at the
initiation of HAART) were significantly higher than those in
subjects with nadirs ⭐200 cells/mm3 (P p .02 ) (figure 1b). We
observed a similar trend with overlapping HIV-1 Gag peptides,
but limited sample availability prevented testing of all the subjects with this panel of peptides (P p .10) (figure 1c).
We next evaluated the relationship between CD4+ T cell nadirs and proliferative responses to HIV-1 p24, a recall antigen
(Candida), and a mitogen (PHA). PHA was used as a positive
control in these assays, and only 1 subject was excluded from
this analysis for having a Dcpm value for PHA of !1000. There
was no relationship between CD4+ T cell nadirs and the ability
to proliferate in response to Candida antigen (figure 2a). Proliferative responses to PHA were the highest of all the stimulants
Figure 2. Association between low CD4+ T cell nadirs and lower HIV-1–specific proliferative responses. a, Candida; b, phytohemagglutinin (PHA);
c, HIV-1 p24 antigen (Protein Sciences); d, HIV-1 IRC p24 protein. White triangles, CD4+T cell nadir ⭐200 cells/mm3; black triangles, CD4+T cell nadir
1200 cells/mm3.
BRIEF REPORT • JID 2006:194 (1 September) • 663
tested, and these responses were significantly higher in subjects
with higher CD4+ T cell nadirs (for subjects with 1200 cells/
mm3, median, 6102.1 Dcpm [interquartile range {IQR}, 4231–
8161 Dcpm]; for subjects with ⭐200 cells/mm3, 3547.1 Dcpm
[IQR, 2344–6394 Dcpm]; P p .02) (figure 2b). Likewise, HIV1–specific proliferative responses to a commercially available
p24 antigen (Protein Sciences) were higher in subjects with
higher CD4+ T cell nadirs (for subjects with 1200 cells/mm3,
481.7 [IQR, 55–1456]; for subjects with ⭐200 cells/mm3, 67
[IQR, 12–663]; P p .05) (figure 2c). We observed similar results
when the IRC p24 antigen (purified from Remune vaccine
immunogen) was used (for subjects with 1200 cells/mm3, 265.0
[IQR, 48–624]; for subjects with ⭐200 cells/mm3, 68.9 [IQR,
5–292]; P p .04) (figure 2d). Results from the 2 separate preparations of p24 were complementary. These data indicate an
effect of CD4+ T cell nadirs on HIV-1–specific T cell responses
in subjects with optimal viral suppression due to antiretroviral
treatment.
In a multivariate analysis, the CD4+ T cell nadir was the most
important independent predictor of the magnitude of HLA
optimal class I peptide responses (P p .008 ) and the level of
p24-specific proliferation for both antigen preparations studied
(Protein Sciences p24, P p .02; IRC p24, P p .02), after we controlled for the duration of antiretroviral treatment and CD4+ T
cell or CD8+ T cell activation status. Duration of HAART was
also independently positively associated with the magnitude of
IFN ELISpot responses (P p .05) and proliferative responses to
both p24 antigens (for Protein Sciences p24, P p .05; for IRC
p24, P p .004). We did not find that the CD4+ T cell count at
the time of the immune assays predicted the magnitude of HIV1–specific immune responses, and the duration of HAART was
not significantly different between groups with low and high
CD4+ T cell nadirs (data not shown).
There was an inverse association between higher levels of T
cell activation and the magnitude of HIV-1 p24–specific proliferation in the overall cohort (CD4+ T cell activation, P p
.04; CD8+ T cell activation, P p .03). However, immune activation was not independently associated with proliferative or
CD8+ T cell epitope-specific responses.
Discussion. We found strong independent associations between CD4+ T cell nadirs and HIV-1–specific immune responses.
Previous studies have evaluated the relationship between CD4+
T cell nadirs and proliferative responses to recall and HIV-1
antigens [13], as well as the response to vaccination [14], and
those researchers concluded that low CD4+ nadirs were associated with a lower degree of immune reconstitution. However,
functional studies were limited to cellular proliferation in response to protein antigens. We found a strong correlation between CD4+ T cell nadirs and CD4+ T cell counts during the
study. This is consistent with data published elsewhere [5].
664 • JID 2006:194 (1 September) • BRIEF REPORT
However, the CD4+ T cell nadir, and not the current CD4+ T
cell count, was an independent predictor of immune reactivity.
These data suggest that subjects with the lowest CD4+ T cell
nadirs have variable recovery of their CD4+ T cell counts and
that, even when there is recovery of CD4+ T cells, HIV-1–
specific immune responses are not completely reconstituted.
We found lower frequencies of IFN-g–secreting cells in response to HLA class I optimal epitopes in subjects with low
CD4+ T cell nadirs. One explanation for this finding is an inability to maintain CD8+ T cell responses when CD4 help is
relatively deficient, which is consistent with the lower levels of
proliferative responses to 2 different HIV-1 p24 antigens found
in subjects with low CD4+ T cell nadirs. However, even among
subjects with low CD4+ T cell nadirs, there were several with
significant responses (Dcpm 1 1000 and corresponding SI 1 10).
Furthermore, there was no clear threshold nadir that predicted
a complete absence of HIV-1–specific immune responses. These
results suggest that, although a low CD4+ T cell nadir may partially predict less-robust helper responses, prolonged HAART
may afford reconstitution even in subjects with CD4+ T cell
nadirs !200 cells/mm3.
We also found a positive association between the duration
of HAART and the magnitude of immune responses. Although
prior longitudinal studies have found decreasing HIV-1–specific
immune responses during HAART [15], our cross-sectional data
suggest that a group of subjects with a longer duration of complete viral suppression may be “enriched” for individuals with
higher magnitudes of HIV-1–specific immune responses. Further studies will be necessary to evaluate the long-term outcome
of subjects with persistent helper and CD8+ T cell responses
during HAART.
The evaluation of immune responses to HIV may be important
to determine the risk of viral breakthrough in future studies of
subjects receiving successful antiretroviral therapy. If the maintenance of virus-specific CD4+ and CD8+ T cell responses is
important for continued viral suppression during HAART, individuals with low CD4+ T cell nadirs may be the most appropriate population for HIV immunotherapy trials.
Acknowledgments
We thank the patients, for their participation in the study; Dirk MeyerOlson, for stimulating discussions and review of the manuscript; Nicole
Grosskopf, Jennifer Nowak, Laura Moran, and Julie Lafalce, for helping us
to collect data; and Drs. Martin S. Hirsch, Mary Albrecht, Paul Skolnik,
Paul Edward Sax, Charles Flexner, Judith A. Aberg, Judith Silverstein Currier, Eric S. Daar, Margaret A. Fischl, Richard C. Reichman, Gene D. Morse,
Kathleen E. Squires, Michael M. Lederman, Mitchell (Mitch) Goldman,
Michael P. Dube, Donna Mildvan, Joseph J. Eron, Thomas B. Campbell,
Pablo Tebas, and William A. O’Brien, for enrolling subjects in the study.
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