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IMMUNOBIOLOGY
Decreases in IL-7 levels during antiretroviral treatment of HIV infection suggest a
primary mechanism of receptor-mediated clearance
Jessica N. Hodge,1Sharat Srinivasula,2Zonghui Hu,3Sarah W. Read,4Brian O. Porter,1Insook Kim,5JoAnn M. Mican,6
Chang Paik,7Paula DeGrange,8Michele Di Mascio,3and Irini Sereti1
1Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, MD; 2Biostatistics
Research Branch, SAIC-Frederick Inc, NCI-Frederick, Frederick, MD; 3Biostatistics Research Branch, NIAID, Bethesda, MD; 4Division of AIDS, NIAID,
Bethesda, MD; 5Applied/Developmental Research Directorate, SAIC-Frederick Inc, Frederick, MD; 6Division of Clinical Research, NIAID, Bethesda, MD;
7Radiopharmaceutical Laboratory, Nuclear Medicine, Radiology and Imaging Sciences, Clinical Center, Bethesda, MD; and 8Battelle/Charles River–Integrated
Research Facility–NIAID Frederick, National Institutes of Health, Bethesda, MD
IL-7 is essential for T-cell homeostasis.
Elevated serum IL-7 levels in lymphopenic
states, including HIV infection, are thought
to be due to increased production by
homeostatic feedback, decreased recep-
tor-mediated clearance, or both. The goal
of this study was to understand how
immune reconstitution through antiretro-
viral therapy (ART) in HIVⴙpatients af-
fects IL-7 serum levels, expression of the
IL-7 receptor (CD127), and T-cell cycling.
Immunophenotypic analysis of T cells
from 29 HIVⴚcontrols and 43 untreated
HIVⴙpatients (30 of whom were followed
longitudinally for <24 months on ART)
was performed. Restoration of both CD4ⴙ
and CD8ⴙT cells was driven by increases
in CD127ⴙnaive and central memory
T cells. CD4ⴙT-cell subsets were not fully
restored after 2 years of ART, whereas
serum IL-7 levels normalized by 1 year of
ART. Mathematical modeling indicated
that changes in serum IL-7 levels could
be accounted for by changes in the recep-
tor concentration. These data suggest
that T-cell restoration after ART in HIV
infection is driven predominantly by
CD127ⴙcells and that decreases of se-
rum IL-7 can be largely explained by
improved CD127-mediated clearance.
(Blood. 2011;118(12):3244-3253)
Introduction
T-cell homeostasis tightly regulates the composition of the T-cell
compartment,1but many aspects of these homeostatic mechanisms
in humans remain unclear. IL-7 is a cytokine produced by stromal
cells in the BM, thymus, and lymph node2and is critical for T-cell
thymopoiesis. Through the JAK-STAT5 signaling pathway, IL-7 is
essential for the development, maturation, and survival of naive
and memory T cells in the periphery.2-6
The IL-7 receptor (IL-7R) consists of 2 chains: the ␣chain
(IL-7R␣/CD127) and the common ␥chain shared by IL-2, IL-4,
IL-9, IL-15, and IL-21.7Previous studies have found that, in
addition to T cells, dendritic cells and monocytes also express
CD127.8,9 Elevated levels of serum or plasma IL-7 have been
observed in CD4 T-cell lymphopenia, including HIV infection,
idiopathic CD4 lymphocytopenia, and after chemotherapy and BM
transplantation.10-13 A strong inverse association has been observed
between serum IL-7 levels and CD4 T-cell counts, especially in
severe CD4 T-cell lymphopenia (⬍200 cells/L).10,11 Two pos-
sible hypotheses have been proposed to explain this association:
increased production of IL-7 as part of a compensatory homeostatic
response (possibly regulated by the presence of CD127 receptor on
dendritic cells) or decreased receptor-mediated clearance from
reduced availability of CD127 in lymphopenic conditions14-17
leading to accumulation of IL-7. It has been previously described
that CD8 T-cell proliferation is more effective than CD4 prolifera-
tion in recovering depleted populations in response to homeostatic
signals.18 Some studies have further suggested that very high IL-7
levels may negatively affect CD4 T-cell homeostatic proliferation,9
although in other animal models of lymphopenia-induced prolifera-
tion, excess IL-7 led to improved restoration of the T-cell pool,
despite high serum levels.19
The goal of our study was to understand how immune reconsti-
tution resulting from combination antiretroviral therapy (ART) and
HIV viral suppression in HIV-infected patients affects longitudinal
CD127 expression and cycling of CD4 and CD8 T cells with the
aim of addressing the fundamental question of IL-7 regulation and
T-cell homeostasis in HIV infection. By mathematical modeling,
we attempted to determine whether changes in serum IL-7 levels
could be attributed to alterations of receptor-mediated clearance.
Methods
Patients
Forty-three HIV-1⫹patients who were enrolled in National Institute of
Allergy and Infectious Diseases (NIAID) Institutional Review Board–
approved protocols at the National Institutes of Health (NIH) Clinical
Research Center were followed between 1996 and 2007. At entry, all
patients signed informed consent in accordance with the Declaration of
Helsinki and were ART naive. Thirty participants with a CD4 T-cell
count ⬍350 cells/L initiated ART and were longitudinally followed for
Submitted December 6, 2010; accepted July 5, 2011. Prepublished online as
Blood First Edition paper, July 21, 2011; DOI 10.1182/blood-2010-12-323600.
Presented in poster form at the 16th Conference on Retroviruses and Opportunistic
Infections, Montreal, QC, February 8-11, 2009; at the Keystone Symposium for “HIV
Immunobiology: from Infection to Immune Control,” Keystone, CO, 2009; and “HIV
Pathogenesis” in Banff, AB, March 27 throughApril 1, 2008.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
3244 BLOOD, 22 SEPTEMBER 2011 䡠VOLUME 118, NUMBER 12
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ⱕ2 years with HIV-1 RNA ⬍500 or ⬍50 copies/mL, depending on assay
sensitivity. PBMCs and serum were collected and cryopreserved before
ART initiation at month 0 (M0) and at months 1, 3, 6, 12, and 24 (M1, M3,
M6, M12, and M24) after ART initiation. HIV-1 RNA levels were
determined with a modification of the Roche Amplicor HIV Monitor assay
kit with a lower limit of detection of 500 or 50 copies/mL. Twenty-nine HIV
seronegative controls (HCs) were also studied after enrollment in a blood
draw NIH Institutional Review Board–approved protocol at the NIH
Clinical Research Center.
Immunophenotyping
Immunophenotypic analysis of cryopreserved PBMCs was performed by
8-color flow cytometry. PBMCs were thawed in RPMI 1640 (Sigma-
Aldrich) supplemented with 10% FCS and DNase I (10 U/mL; Roche
Diagnostics). PBMCs were washed in PBS (Invitrogen), and dead cells
were stained (Live/dead Fixable Blue Dead Cell Stain Kit; Invitrogen).
Surface staining was performed with the following monoclonal antibodies:
CD4-QDot 605 (Invitrogen), CD8-Pacific Blue (R&D Systems), CD45RO-
Cy7PE (BD), CD27-APC (BD Biosciences), and CD127-PE (Beckman
Coulter). PBMCs were then washed, fixed, and permeabilized (Fix/Perm;
eBioscience) according to the manufacturer’s instructions. Intracellular
staining for the nuclear antigen Ki67 was performed (Ki67-FITC; BD
PharMingen).
Samples were collected on an LSR II (BD Biosciences) with the use
of FACS Diva software. Approximately 4 ⫻105total events were
collected per sample. Flow cytometric data were analyzed with FlowJo
Version 8 software (TreeStar). Viable CD3⫹cells were selected, and then
events were sequentially gated on CD4⫹or CD8⫹T cells and then on naive
and memory populations. Naive cells were defined as CD45RO⫺CD27⫹.
Memory cells were defined as CD45RO⫹CD27⫹(central memory; CM) or
CD45RO⫹CD27⫺(effector memory; EM). Afourth population of cells was
defined as CD45RO⫺CD27⫺(effectors).
Cytokine values
Serum IL-7 levels in cryopreserved sera were measured with the Quantikine
high-sensitivity human IL-7 immunoassay (R&D Systems) with a lower
limit of detection ⬍0.1 pg/mL. The assay was performed according to the
manufacturer’s instructions, and all specimens were run in duplicate.
[125I]IL-7 binding assays
The binding affinity of IL-7 to its receptor (CD127) and the number of
CD127 receptors per cell were estimated through an [125I]IL-7 binding
assay with the use of freshly isolated PBMCs from 2 healthy donors.
Recombinant human IL-7 was kindly provided by Cytheris. Iodination of
IL-7 was performed with [125I]NaI with the use of the Iodogen method as
previously described.20 [125I]IL-7 was purified by a size exclusion PD-10
column eluted with 0.02M sodium phosphate and 0.155M sodium chloride
(normal saline), pH 7.2. The purified [125I]IL-7 was stored at 4°C in 0.02M
sodium phosphate to 0.155M sodium chloride (normal saline solution), pH
7.2, containing 2% BSA and had a specific activity of 48 Ci (1.8 Bq)/g.
After PBMC isolation, erythrocytes were lysed with ACK lysing buffer
(BioWhittaker). Binding assays were performed as previously described.21
Briefly, PBMCs were washed and resuspended in the binding medium at a
concentration of 9 ⫻106cells/mL in PBS containing 2% BSA, 20mM
HEPES buffer, and 0.2% sodium azide at pH 7.2. Total binding was
measured by incubating cells at various concentrations of [125I]IL-7
[0.05-10nM] for 30 minutes at 37°C. Nonspecific binding was measured by
first incubating cells with ⱖ1000-fold molar excess of unlabeled IL-7 for
20 minutes at 37°C and subsequently with various concentrations of
[125I]IL-7 for 30 minutes at 37°C. After incubation, samples were centri-
fuged for 2 minutes at 12 000 rpm, and the counts per minute in the cell
pellet were measured. All data were corrected for nonspecific binding. The
equilibrium binding data were fitted to an equation consisting of the sum of
2 Michaelis-Menten terms22 with the use of the nonlinear least-squares
Levenberg-Marquardt algorithm.23 The 95% confidence interval of the
estimates was obtained by bootstrapping the residuals between the observed
data and the best-fitted (theoretical) values.
Statistical analysis
Wilcoxon rank-sum test was used for comparison between HC and HIV⫹
(n ⫽30) at each time point, and Wilcoxon signed-rank test was used for
paired comparisons between each on-ART time point and M0. Association
between 2 variables was explored by Spearman rank correlation coefficient
(R) and simple linear regressions. Association between one variable and
multiple covariates was explored by multiple linear regressions, after
log-transformation of viral load and proliferation data; for these analyses,
one patient was eliminated because of the inability to measure levels of
proliferation (below the limits of the assay). To account for the multiple
tests, differences or associations with P⬍.01 were considered statistically
significant.
In the longitudinal analysis, the growth rate of cell populations after M3
was calculated as the change in cell count between 2 adjacent time points
divided by the change in time between the 2 time points; the growth rate
before M3 was calculated as the regression slope among the 3 measure-
ments at M0, M1, and M3. The rate of change of cell populations or IL-7
level was also estimated by nonparametric local linear mixed-effects
modeling (24 and supplemental Methods, available on the Blood Web site;
see the Supplemental Materials link at the top of the online article). At any
time t, the longitudinal curve of the variable was locally approximated by a
line, with the slope of the line giving the rate of change at t. Letting tvary
over the observation period, we obtained the time profile for the rate of
change in the variable. Applying nonparametric local linear mixed-effects
modeling simultaneously to a pair of variables, we obtained the time profile
for the correlation between the rates of change in the 2 variables.25
Mathematical modeling
A mathematical model was used to test whether changes in serum IL-7
levels during ART could be explained by receptor-mediated clearance on
the basis of CD127 expression on T cells. The model was based on the
idealization of an in vitro experiment in which the binding of IL-7 ligand to
the surface CD127 receptors is governed by the law of mass action coupled
to a first-order kinetics of ligand-induced internalization26 and is explained
by 3 differential equations for the unbound ligand (L), unoccupied receptors
(R), and the complex (C; ie, the receptors occupied by the ligand). The rates
of association kf, dissociation kr, and internalization ke, are constants and
depend only on the type of ligand and receptor. The generalization of the
model for the in vivo dynamics of the interplay between IL-7 serum levels
and changes in CD127 receptors assumes that the ligand (L) is produced and
cleared from the peripheral system at constant rates aand cl, respectively,
and the receptors (R) are refilled at a constant source rate sand grow and
disappear at rates p(t) and d(t), respectively. The receptors occupied by the
ligand, C, like the unbound receptors, also disappear at rate d(t). The rates
of growth and disappearance for the receptors (R) will change from their
quasi steady state values at baseline to reach a new quasi steady state level
after initiation of ART. The cell binding assays performed on fresh PBMCs
from 2 healthy donors confirmed previous observations from other groups
that CD127 receptors display dual affinity binding to IL-721,27; thus, the
aforementioned differential equations for the receptors (R) and the complex
(C) will be translated in the following 5 differential equations to account for
high- and low-affinity receptors, where R ⫽R1 ⫹R2 and C ⫽C1 ⫹C2.
dL
dt
⫽a⫺clL ⫺kfLR ⫹krC
dRi
dt
⫽si⫹pRi⫺dRi⫺kfiLRi⫹kriCii⫽1,2 (1)
dCi
dt
⫽kfiLRi⫺kriCi⫺dCi⫺keCii⫽1,2
Data analysis. The coupled set of differential equations were solved
numerically using the “Runge-Kutta 4” ODE solver, that is, a fixed
step-size, single-step explicit Runge-Kutta fourth-order method. Curve
IL-7 LEVELS IN TREATED PATIENTS WITH HIV 3245BLOOD, 22 SEPTEMBER 2011 䡠VOLUME 118, NUMBER 12
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fitting, confidence interval estimates, and model simulations were per-
formed with Labview 2010 (National Instruments). Steady state solution
was solved with Mathematica 6 (Wolfram Research). Statistical analyses
were performed with R software (R Project).
Results
Clinical characteristics
The baseline clinical characteristics of the ART-naive HIV⫹
patients (n ⫽43), including the subgroup that received ART, and
HCs are shown in Table 1. Longitudinal graphs include only those
patients who initiated ART after their baseline visit (n ⫽30).
By M6, 76% of HIV⫹patients (22 of 29) achieved plasma
viremia below detection levels (viral load data were missing for
one patient at M6, but suppression was documented at M12). HIV⫹
patients had significantly lower CD4⫹and higher CD8⫹T cells/L
and higher serum IL-7 levels than HCs at most time points (Figure
1A-B,D; significant Pvalues are shown as gray symbols). In a
paired comparison of each time point to M0, CD4⫹T-cell count
significantly increased at all time points (Figure 1A; paired
comparison significant Pvalues are represented as black symbols).
CD4⫹T cells/L and IL-7 levels were significantly inversely
associated in HIV⫹patients at baseline (R⫽⫺0.76, P⬍.0001;
Figure 1C) but not in the HC group (R⫽⫺0.34, P⫽.068). HCs
were significantly older than the HIV⫹patients (Table 1), but there
was no association between age and serum IL-7 levels in the HCs
or the HIV⫹patients (supplemental Figure 1). IL-7 levels de-
creased from M1 onward and did not differ from HC levels at M12
and beyond (Figure 1D). In addition, HIV⫹patients had a
significantly lower proportion of CD4⫹CD127⫹and CD8⫹CD127⫹
T cells than HCs, with restoration in the CD8⫹but not in the CD4⫹
T-cell subset at M24 (Figure 1E-F). The proportion of CD4⫹and
CD8⫹T cells expressing CD127 began increasing significantly
compared with baseline at M6 and beyond (Figure 1E-F).
Cycling in T-cell subsets
Although CD8⫹T cells initially showed increased cycling (Ki67⫹)
that normalized by M3, CD4⫹T cells maintained increased cycling
compared with HCs up to M6 (Figure 2A-B). The proportion of
cycling CD3⫹T cells was also increased but normalized by M3
(Figure 2C). Similarly, increased cycling of both CD3⫹CD127⫹
and CD8⫹CD127⫹cells normalized shortly after ART initiation
compared with HCs, whereas increased cycling in CD4⫹CD127⫹
T cells was observed through M6 (Figure 2D-F). The cycling of
CD4⫹CD127⫺and CD8⫹CD127⫺T-cell subsets was not different
from the HCs at most time points studied (Figure 2G-I).
The proportion of cycling CD4⫹, CD4⫹CD127⫹, and
CD4⫹CD127⫺T cells did not significantly change from M0 to M1
and M3 but decreased at M6, M12, and M24. The proportion of
cycling CD8⫹and CD8⫹CD127⫹T cells decreased from M0 at M3
and at all time points thereafter. The proportion of cycling CD3⫹,
CD3⫹CD127⫹, and CD3⫹CD127⫺T cells decreased from M0 at
M6, M12, and M24 (Figure 2).
Univariate linear regressions showed a positive association of
the percentage of proliferating CD4⫹CD127⫹T cells with HIV-1
RNA (slope ⫽0.42, P⫽.019) and serum IL-7 (slope ⫽0.02,
P⫽.027) levels and an inverse association with the CD4⫹CD127⫹
T-cell counts (slope ⫽⫺0.0072, P⬍.001) at baseline (M0) and
similarly at M1. However, the multivariate linear regression
involving all the above covariates showed a significant association
only between the percentage of proliferating CD4⫹CD127⫹T cells
and total number of CD4⫹CD127⫹T cell/L(P⬍.01 ⱕ1 year
and P⫽.023 at 2 years on ART).
The numbers of cycling CD4⫹, CD8⫹, and CD3⫹T cells are
shown in supplemental Figure 2. The number of cycling CD4⫹
T cells was similar, but the numbers of cycling CD8⫹and CD3⫹
T cells were higher compared with HCs (supplemental Figure
2A-C). The number of cycling CD127⫹T cells was similar to
controls in the CD8⫹and CD3⫹subsets yet was significantly lower
in the CD4⫹T-cell subsets (supplemental Figure 2D-F).
Recovery of T cells expressing CD127
In the HIV⫹group, CD4⫹CD127⫹, CD8⫹CD127⫹, and
CD3⫹CD127⫹T cells/L were significantly lower than HCs at M0
(28 vs 485 T cells/L, P⬍.0001; 71 vs 157 T cells/L, P⫽.034;
and 112 vs 641 T cells/L, P⬍.0001, respectively) and increased
over time (Figure 3A-C). In contrast, CD4⫹CD127⫺, CD8⫹CD127⫺,
and CD3⫹CD127⫺T cells/L increased from M0 to M1 and then
leveled off. CD4⫹CD127⫹T cells increased continuously through-
out ART but did not normalize by M24 (Figure 3A). In contrast,
CD8⫹CD127⫹T cells normalized by M1 but then exceeded HC
levels at both M12 and M24 (Figure 3B). At all study time points
(except M12) there was a significant positive correlation between
CD4⫹CD127⫹and CD8⫹CD127⫹T cells/L (overall r⫽0.53,
P⬍.0001). The number of CD3⫹CD127⫹T cells/L increased
with ART and reached HC levels by M24 (Figure 3C). Compared
with M0, T-cell counts increased at M1 (CD4⫹CD127⫹,
CD4⫹CD127⫺, CD8⫹CD127⫹, and CD3⫹CD127⫺cells/L) and
at M3 (CD3⫹CD127⫹cells/L) from paired comparisons within
the HIV⫹group.
CD127 expression on naive and CM T-cell subsets
Restoration of total numbers of CD4⫹and CD8⫹naive, CM, EM,
and effector T cells/L were studied (supplemental Figure 3). The
number of naive and CM CD4⫹T cells did not reach HC levels,
whereas CD8⫹naive T cells normalized, and CM cells were higher
than HCs (supplemental Figure 3A-D). Effector memory and
Table 1. Baseline clinical characteristics of all ART-naive HIVⴙstudy participants, the subset of HIVⴙparticipants that initiated ART, and
HIV-seronegative controls (HCs)
HIVⴙ(n ⴝ43), median
(interquartile range)
HIVⴙ/ART (n ⴝ30), median
(interquartile range)
HC (n ⴝ29), median
(interquartile range)
P
HIVⴙvs HC HIVⴙ/ART vs HC
Age, y 38 (33-43) 36 (33-42) 47 (43-58) .0002 .0002
CD4⫹T cells/L187 (47-383) 69 (21-229) 623 (432-879) ⬍.0001 ⬍.0001
CD4⫹T cells, % 15 (5-26) 8 (2-19) 45 (40-52) ⬍.0001 ⬍.0001
CD8⫹T cells/L539 (351-977) 457 (290-798) 291 (230-470) .0005 .023
CD8⫹T cells, % 58 (45-66) 60 (49-68) 22 (17-30) ⬍.0001 ⬍.0001
Serum IL-7, pg/mL 24.8 (19.3-36.3) 30.0 (22.3-40.5) 15.3 (12.3-19.6) ⬍.0001 ⬍.0001
Log10 plasma HIV-1 RNA, copies/mL 4.76 (3.98-5.15) 5.02 (4.48-5.41) NA NA NA
3246 HODGE et al BLOOD, 22 SEPTEMBER 2011 䡠VOLUME 118, NUMBER 12
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effector CD4⫹T cells quickly normalized, whereas EM and
effector CD8⫹T cells were higher than HCs (supplemental Figure
3E-H). The number of CD4⫹T-cell memory subsets increased
significantly from M0 to subsequent time points. Only naive CD8⫹
T cells increased significantly from M0 to most studied time points.
CD127 expression on naive CD4⫹T cells increased from M0
throughout ART but did not normalize by M24 (Figure 4A). In
contrast, the number of naive CD4⫹CD127⫺T cells/L reached
levels similar to HC by M12 (Figure 4C). CD127 expression on
naive CD8⫹T cells also increased with ART but exceeded levels
seen in HCs at both M12 and M24 (Figure 4B). The CD4⫹CM
T-cell subset closely paralleled the naive subset (Figure 4E,G). The
number of CD8⫹CD127⫹CM T cells/L was low at M0 but
normalized subsequently (Figure 4F). The number of CD8⫹CD127⫺
CM T cells/L exceeded HC levels at all time points (Figure 4H).
The number of naive CD8⫹CD127⫺T cells/L never differed from
HCs (Figure 4D). In the paired comparisons, naive CD4⫹CD127⫹,
CD4⫹CD127⫺, CD8⫹CD127⫹and CM CD4⫹CD127⫹,
CD4⫹CD127⫺, CD8⫹CD127⫹all increased at M1 or M3 com-
pared with M0 (Figure 4A-C,E-G).
Growth rate of CD127ⴙand CD127ⴚsubsets in CD4ⴙand
CD8ⴙT cells
The growth rate of various cell populations is graphically depicted
in Figure 5C to L. The growth rate of the CD127⫹subsets was
higher than the growth rate for the CD127⫺subsets, particularly at
later time points (M6 and beyond), in both CD4⫹and CD8⫹T cells
once the HIV viral load was suppressed. However, this trend held
true only for naive and CM cell populations and not for EM and
effector cell subsets.
Analysis of the model
Extracellular (free) ligand (L) binds reversibly to free surface
receptor (R) with an on rate kfand an off rate krto form the
ligand-receptor complexes (C). The ligand-receptor complexes are
endocytosed with an average rate constant ke. The free ligand enters
Figure 1. CD4ⴙand CD8ⴙT cells/L and proportion of CD4ⴙCD127ⴙT cells do not normalize with ART,whereas IL-7 levels do not differ from HCs after 12 months of
ART.Data shown include the total number of CD4⫹(A) and CD8⫹(B) T cells/L, serum IL-7 pg/mL (D), and proportion of CD4⫹CD127⫹(E) and CD8⫹CD127⫹(F) T cells in the
group of HIV⫹patients (n ⫽30) who were followed longitudinally from M0 to M24. The association between CD4⫹T cells/L and serum IL-7 levels in HIV⫹at M0 and HCs is
shown (C). Circles (black) represent median values and vertical bars indicate interquartile range (IQR). Gray dashed lines indicate HC median values. Pvalues (gray) at the top
of the graphs represent unpaired comparisons between HC and HIV⫹at each time point, and Pvalues (black) above the IQR bars indicate paired comparisons of HIV⫹patients
at each time point during the therapy to the pretherapy level (M0). Significant Pvalues ⬍.01 are reported, *P⫽.01 ⬎.001, **P⫽.001 ⱖ.0001, and ***P⬍.0001. HIV⫹data
are shown as black circles and HCs as gray squares.
IL-7 LEVELS IN TREATED PATIENTS WITH HIV 3247BLOOD, 22 SEPTEMBER 2011 䡠VOLUME 118, NUMBER 12
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the compartment at a rate aand is cleared at a rate cl. The
peripheral system is continuously replenished with new receptors
at a constant rate s(time⫺1). Receptors grow in number at a rate p.
Both unbound and bound receptors (C) also disappear at a rate d.
As described in “Methods,” applying the law of mass action to
this compartmental scheme generates a system of 5 differential
equations that accounts for the high- and low-affinity displayed by
the CD127 receptors to its ligand, IL-7.
The binding affinity of IL-7 to the IL-7 receptor (CD127) and
the number of CD127 receptors per cell were estimated through a
[125I]IL-7 binding assay with the use of fresh PBMCs from 2
healthy donors. As shown in the insert plots in Figure 6, the
Figure 2. Increased proportion of cycling (Ki67ⴙ) CD4ⴙand CD4ⴙCD127ⴙT cells persists up to M12 after ART, whereas the proportion of cycling CD8ⴙand
CD8ⴙCD127ⴙT-cell normalizes shortly after ART initiation. The proportion of cycling CD4⫹(A), CD8⫹(B), CD3⫹(C), CD4⫹CD127⫹(D), CD8⫹CD127⫹(E), CD3⫹CD127⫹
(F), CD4⫹CD127⫺(G), CD8⫹CD127⫺(H), and CD3⫹CD127⫺(I) is shown in the group of HIV⫹patients (n ⫽30) who were followed longitudinally from M0 to M24. Circles
(black) represent median values, and bars indicate interquartile range (IQR). Gray dashed lines indicate HC median values. Pvalues (gray) at the top of the graphs represent
unpaired comparison between HC and HIV⫹at each time point, and Pvalues (black) above the IQR bars indicate paired comparison of HIV⫹patients at each time point during
the therapy to the pretherapy level (M0). Significant Pvalues ⬍.01 are reported, *P⫽.01 ⬎.001, **P⫽.001 ⱖ.0001, and ***P⬍.0001.
Figure 3. Restoration of CD4ⴙ, CD8ⴙ, and CD3ⴙT cells/L expressing CD127 after ART initiation. The total number of CD4⫹CD127⫹(A), CD8⫹CD127⫹(B),
CD3⫹CD127⫹(C), CD4⫹CD127⫺(D), CD8⫹CD127⫺(E), and CD3⫹CD127⫺(F) T cells/L is shown in the group of HIV⫹patients (n ⫽30) who were followed longitudinally
from M0 to M24. Circles (black) represent median values, and bars indicate interquartile range (IQR). Gray dashed lines indicate HC median values. Pvalues (gray) at the top
of the graphs represent unpaired comparison between HC and HIV⫹at each time point, and Pvalues (black) above the IQR bars indicate paired comparison of HIV⫹patients at
each time point compared with the pretherapy level (M0). Significant Pvalues ⬍.01 are reported, *P⫽.01 ⬎.001, **P⫽.001 ⱖ.0001, and ***P⬍.0001.
3248 HODGE et al BLOOD, 22 SEPTEMBER 2011 䡠VOLUME 118, NUMBER 12
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Scatchard analysis of the data in both binding experiments yielded
a curvilinear plot, confirming that CD127 receptors display dual-
affinity binding to IL-7, as previously reported.21,27 The high-
affinity receptors had a Kdof 0.155nM (95% CI, 0-0.52nM) and
0.112nM (95% CI, 0-0.52nM) and 678 (95% CI, 265-1378) and
588 (95% CI, 241-1368) CD127 receptors per cell, respectively,
with kd⫽kr/kf; the low-affinity receptors had a Kdof 9.84nM (95%
CI, 5.8-22.97nM) and 11.56nM (95% CI, 5.63-72.5nM) and 9058
(95% CI, 7321-13 944) and 7608 (95% CI, 5607-27 003) CD127
receptors per cell, respectively. Thus, ⬃7% of the CD127 recep-
tors display high-affinity binding.
The following parameters and assumptions were used in
simulating the model described in equation 1: an estimate for the
clearance rate of IL-7 (cl) is obtained from the decay in plasma
levels observed after administration of rhIL-7 in HIV-1 infected
patients.28 In that study, a clearance of 1 log in ⬃20 hours was
observed, which translates into a clearance rate (cl) of 0.115
hour⫺1. An estimate for the endocytosis rate, ke, of 0.1467 hours⫺1,
is provided from the kinetics of the percentage of internalized
CD127-bound receptors described in Carini et al.29 The receptor
growth and disappearance rates, pand d, respectively, at steady
state, are inferred from the proliferation and disappearance rates as
estimated from in vivo labeling studies in HIV-infected patients,30
because the rates of change in receptors can be assumed to equal
the rate of changes in CD127⫹T cells. The association and
dissociation rates are unknown from our study, because only their
ratio could be estimated from the [125I]IL-7 binding assay; how-
ever, simulations showed low sensitivity to changes in kror kf,
whereas the dynamics of IL-7 changes are mostly dominated by
their ratio (data not shown). The values of rate of free ligand
entering the compartment (a), the source rate of receptors (si), and
the baseline ligand-receptor complexes (Ci) were obtained from the
steady state equations.
The model was simulated (in time scale of hours) for 720 days
of ART therapy. The initial values of free ligand and free receptors
and the fold-changes were obtained from the mean levels of serum
IL-7 and mean number of CD127⫹receptors from the 30 HIV⫹patients
who underwent therapy. The mean serum IL-7 level was 33.8 pg/mL
(1.35 ⫻10⫺12M) at M0 and 18.7 pg/mL (7.47 ⫻10⫺13M) at M24
(IL-7 MW ⫽25 kDa). The mean number of CD127⫹T cells in the
blood was 201/L and 578/L at M0 and M24, respectively, which
converts to receptor molar concentrations of 2.99 ⫻10⫺12MatM0
and 8.6 ⫻10⫺12M at M24. The receptor molar concentration was
calculated from the total number of CD127 receptors estimated
from the binding assay (8966/cell). The initial values for ligand-
receptor complexes were obtained from the steady state solution.
We used baseline (M0) steady state levels of growth and disappear-
ance rates P⫽.001 hour⫺1and d⫽0.004 hour⫺1, respectively.
Moreover, the growth rate [p(t)] during the course of the therapy is
assumed to follow the observed pattern in proportion of proliferat-
ing CD127⫹T cells as inferred from the Ki67 staining (linear
interpolation between 2 adjacent time points), whereas the disap-
pearance rate [d(t)] is assumed to normalize from its pretherapy
steady state level after an exponential decay with a minimum
Figure 4. Restoration of naive and CM T cells expressing CD127
after ART.The total number of naive (A-D) and CM (E-F) CD4⫹CD127⫹,
CD8⫹CD127⫹, CD4⫹CD127⫺, and CD8⫹CD127⫺T cells/L is shown in
the group of HIV⫹patients (n ⫽30) who were followed from M0 to M24.
Circles (black) represent median values and bars indicate interquartile
range (IQR). Gray dashed lines indicate HC median values. Pvalues
(gray) at the top of the graphs represent unpaired comparisons between
HC and HIV⫹at each time point, and Pvalues (black) above the IQR
bars indicate paired comparison of HIV⫹patients at each time point
during the therapy to the pretherapy level (M0). Significant Pval-
ues ⬍.01 are reported, *P⫽.01 ⬎.001, **P⫽.001 ⱖ.0001, and
***P⬍.0001. Naive cells were defined as CD45RO⫺CD27⫹;CMas
CD45RO⫹CD27⫹.
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threshold (fixed as posttherapy disappearance rate observed in
patients treated with ART)30 of 3-fold lower compared with before
therapy (Figure 7). The model was evaluated and updated with
Runge-Kutta fourth-order continuous solver with a step size of
0.01 hour. With the use of the above-mentioned parameters, the
model described in equation 1 shows that ⬍0.1% of total receptors
(CD127) are occupied by the ligand at any time during the course
of therapy (not shown).
Model simulations with the use of the concentration of CD127⫹
receptors in the blood show that the 2.8-fold increase of the CD127
receptors observed in the peripheral blood after 2 years of ART is
not enough to reduce the free ligand concentration from the serum.
However, most lymphocytes reside in the lymphoid tissues (lymph
nodes and spleen), where lymphocytes are highly concentrated.
With the use of the estimate that 70% of 2 ⫻109lymphocytes/g of
lymphoid tissue in HCs are CD3⫹,31 (72% of which are CD127⫹,
and normalizing on the CD127⫹T-cell count in the blood at M0),
the same model shows that a 2.8-fold increase of CD127 receptors
in the lymphoid tissue is enough to decrease the free ligand
concentration in the serum by 2.2-fold (Figure 7).
Our model also predicts an inverse association between the
rates of change of IL-7 and CD127 receptors. Because these rates
may change over time, instead of using linear regressions for
constant rates, the nonparametric local linear mixed-effects model-
ing was applied for estimating the rates of change of IL-7 and
CD127 as functions of time, as well as the correlation between the
2 rates. Figure 5A shows the correlations on the basis of the group
of HIV⫹patients who started ART: the correlation between the rate
of change of IL-7 levels and that of CD3⫹CD127⫺population is
close to zero and is nearly constant over time, whereas a negative
correlation is observed between that of IL-7 levels and
CD3⫹CD127⫹population immediately after ART initiation. The
same trend of correlation is also observed for the CD4⫹T cells
(which predominantly express the CD127 receptor) but not for the
CD3⫹or CD8⫹T cells (Figure 5B).
Discussion
In this HIV⫹cohort treated with ART for 2 years, we found that
CD4⫹T-cell subsets were not completely reconstituted and main-
tained elevated cycling longer in comparison to CD8⫹T-cell
subsets. The restoration of both CD4⫹and CD8⫹T cells was
predominantly driven by increases in CD127⫹naive and CM
T cells. Despite the persistence of low CD4⫹T-cell counts, serum
IL-7 levels normalized by 1 year of ART, suggesting that tight
homeostatic feedback may not significantly contribute to increases
in IL-7 during lymphopenia. These data, in conjunction with
mathematical modeling, suggest that the observed fluctuations of
IL-7 after ART may be related to improved CD127-mediated
clearance as T cells expressing CD127 are restored.
Figure 5. Rates of change of IL-7 and CD127 receptors and growth rates of CD127ⴙand CD127ⴚT cells of CD4ⴙand CD8ⴙT-cell subsets. (A) Time profiles for the
correlation between the rate of change in serum IL-7 levels and that in CD3⫹127⫹T cells/L (red line), and the correlation between the rate of change in serum IL-7 and that in
CD3⫹127⫺(blue line), with the dashed lines indicating the respective 95% confidence intervals. (B) Time profiles for the correlation between the rate of change in serum IL-7
with that in total CD3⫹(black), CD4⫹(red), and CD8⫹(green) T cells/L. The dashed lines represent the 95% confidence intervals. These time profiles are estimated up to
6 months, because later observations were sparse and the algorithm failed to converge. The growth rates of total (C-D), naive (E-F), CM (G-H), EM (I-J), and effector (K-L)
CD4⫹and CD8⫹T cells after ART initiation. Median values are shown. The growth rate from M0 to M3 with intermediate data at M1 is calculated with the linear interpolation and
is plotted at 1.5 months on the x-axis. Pvalues represent the differences in the growth rate between CD127⫹and CD127⫺T cells. Significant Pvalues ⬍.01 are reported,
*P⫽.01 ⬎.001, **P⫽.001 .0001, and ***P⬍.0001.
3250 HODGE et al BLOOD, 22 SEPTEMBER 2011 䡠VOLUME 118, NUMBER 12
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Many studies have previously reported that initiation of ART in
HIV⫹patients decreases T-cell cycling and increases naive T-cell
restoration and CD127 expression in T cells.17,32-35 In our study all
these variables were tested in concert and followed longitudinally
with early post-ART time points to address specifically whether
changes in the T cells expressing the IL-7 receptor could account
for changes in serum IL-7 levels. The idea that IL-7 levels are
elevated as a compensatory response to lymphopenia has much
support, although the mechanism in humans (especially regarding
IL-7 production) is not entirely clear. Napolitano et al found
increased IL-7 in lymphocyte-depleted lymph nodes compared
with hyperplastic specimens with the use of immunohistochemistry
methods.10 However, this could have been because of the local
accumulation of the cytokine in lymphopenic areas. Supranormal
IL-7 levels were found in “CD4-exploders,” HIV⫹people with
very strong responses to ART,36 suggesting that CD4 T-cell count is
significantly influenced by homeostatic control by IL-7. In a mouse
model of lymphopenia CD4⫹T-cell homeostatic proliferation was
regulated through CD127 signaling on antigen presenting cells,9
and high IL-7 levels, either through lymphopenia or exogenous
administration, actually decreased IL-7 production in stroma-
derived cells and in APCs. Although this mechanism of controlling
homeostatic proliferation through decreased IL-7 production was
nicely proven in mice, we do not have adequate means of
measuring IL-7 production in these cell types in humans.
With our compilation of all the relevant variables from a
longitudinal cohort and novel mathematical model we show that
there is consistency, although nonexclusively, with one of the
2 theories about increased IL-7 levels in lymphopenia. On the basis
of the binding affinity of IL-7 for CD127 and the estimated number
of receptors per unit mass of lymphoid organs, the concentration of
this and probably other cytokines measured in the serum, appears
to fall in the right order of magnitude to detect changes in the
concentrations of its receptor. We saw that fluctuations in IL-7
levels during improvement of lymphopenia are less tightly depen-
dent on CD4⫹T-cell counts and more dependent on receptor-
mediated clearance.
The model may not be comprehensive in considering all factors
affecting the clearance of IL-7. Collagen deposition in the lym-
phoid tissue of HIV⫹patients may be ⬃4- to 10-fold higher than in
the uninfected person.37 There are no data about fibronectin
concentrations or changes during ART; however, in vitro binding
assays suggest ultra-low affinity of IL-7 to fibronectin (⬃100- and
10 000-fold lower compared with the 2 types of affinities observed
in our PBMC in vitro data).38 On the basis of our modeling, the
changes in CD127 receptors are indeed sufficient to explain the
decrease in IL-7 observed during therapy, but further investigations
will be needed to address the role of fibronectin or specific types of
collagen in the clearance of free IL-7. In addition, peripheral blood
was exclusively used in our study, but in SIV-infected macaques it
has been clearly shown that CD127 expression in T cells tightly
correlates between tissues and blood.39
Although it is agreed that IL-7 levels are increased in untreated
HIV infection, there is no consensus as to whether they return to
normal levels with effective ART. We found increased IL-7 levels
that normalized at 1 year of ART similar to others,40,41 including a
recent large longitudinal study.42 However, lack of normalization of
serum IL-7 with ART has also been reported,17,43 but the ART-
treated patients were not virologically suppressed or the observed
IL-7 levels were much lower than in our cohort. In most studies though,
including animal models,15 a clear association has been seen
between CD4⫹CD127⫹T cells with changes in serum IL-7 level.
Other mechanisms may also contribute to high IL-7 levels or
may affect the responsiveness of cells to IL-7 in lymphopenia. The
response to IL-7 may depend on thymic function and age.
Reconstitution of naive T cells with ART is decreased in older
HIV⫹patients,44 and a relation between T-cell counts and IL-7 in
patients with low thymic volume has been observed.45 It has also
been shown that T-cell responsiveness to IL-7 may be impaired by
HIV infection34 because of overstimulation and desensitization of
the pSTAT5 signaling pathway. However, the near absence of
viremia at M6 of ART and beyond would suggest that normal
responses to IL-7 should be restored, yet we found persistently
decreased CD4⫹T-cell count with normalized IL-7 levels at 1 and
2 years on ART.
Serum IL-7 levels did not independently correlate with T-cell
cycling. In fact, a transient increase in CD4⫹and CD8⫹T-cell
cycling despite lowering IL-7 levels was seen at M1, suggestive of
improved survival of cycling T cells. In addition, increased cycling
of CD4⫹T cells and CD4⫹CD127⫹cells was maintained longer
than in the CD8⫹T cells even after viral suppression, suggesting
the possibility of lineage-specific proliferative response to lym-
phopenia. Data reported recently46 clearly showed lineage-specific
T-cell homeostatic proliferation in sooty mangabeys and rhesus
macaques after either CD4⫹or CD8⫹T-cell depletion. This is also
in agreement with the observation that different stimuli may be
driving the cycling of CD4⫹(lymphopenia) and CD8⫹(viremia)
T cells47 in HIV. It has been suggested that CD8 recovery in
response to homeostatic proliferation may be more effective than
CD4 recovery and, in fact, that very high IL-7 levels may even
hinder CD4⫹T-cell recovery.9Although CD8⫹T cells express
Figure 6. Equilibrium binding of [125I]IL-7 to fresh PBMCs from 2 healthy
donors. The inserts show Scatchard representation of specific binding; r ⫽molecules/
cell bound.
IL-7 LEVELS IN TREATED PATIENTS WITH HIV 3251BLOOD, 22 SEPTEMBER 2011 䡠VOLUME 118, NUMBER 12
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much lower levels of CD127 than CD4⫹T cells, they can be
responding to other cytokines, including IL-2 and IL-15. In our
study we also found evidence of the preferential recovery of
CD127⫹naive and CM subsets in both CD4⫹and CD8⫹T cells
and corroborated findings of CD4⫹T-cell recovery that lags behind
that of CD8⫹T cells. However, the lack of restoration of
CD4⫹CD127⫹cell numbers (and also CM and naive subsets) and
the increase above baseline levels of the number of CD8⫹CD127⫹
and naive CD8⫹CD127⫹T cells, along with a normalization of the
number of CD3⫹CD127⫹T cells/L by M24 could be indicative of
blind homeostasis that has been shown in mouse48 and BM
transplantation studies49 and has both been supported and refuted in
HIV infection.50 The fact that CD4⫹T cells had not normalized and
yet IL-7 levels and cell cycling decreased by M12 to M24 would
suggest that the subset-specific homeostatic forces may be more
evident and efficient in extreme lymphopenia. It is possible that the
duration of lymphopenia and the relative changes of specific T-cell
subsets that express cytokine receptors, and thus regulate available
levels of circulating cytokines, could be the determinants of
whether homeostasis would appear blind or lineage specific.
In conclusion, we provide evidence that receptor-mediated
clearance could account for changes in IL-7 during reconstitution
of T cells after initiation of ART. These findings offer support for
the involvement of a more complex regulation of T-cell homeosta-
sis in human conditions of severe lymphopenia that goes beyond a
simple IL-7 feedback production loop.
Acknowledgments
The authors thank the patients for their generous donation of
time and study samples as well as the outpatient clinic 8 staff at
NIH who managed their care. They also thank Dr Scott Durum
for helpful discussions and Dr Dean Follmann for statistical
assistance.
This work was supported in part by the Intramural Research
Program of NIAID/NIH.
Authorship
Contribution: I.S., M.D.M., and S.W.R. designed research; I.S. and
J.M.M. provided study samples; J.N.H., S.S., S.W.R., P.D., and
M.D.M. performed research experiments and collected data; S.S.,
Z.H., and M.D.M. performed statistical analysis; I.S., M.D.M.,
S.S., and J.N.H. interpreted and analyzed data; S.S. and M.D.M.
completed mathematical models and analysis; and J.N.H., I.S.,
M.D.M., and S.S. wrote the manuscript. All authors reviewed and
contributed to the manuscript preparation.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Figure 7. Simulation of the model described in equation 1 for
up to 24 months of ART. Theoretical curves predicted by the
model (solid line), for the mean values of the concentration of
receptors (CD127) and free ligand (serum IL-7 levels) in the group
of 30 HIV⫹patients at each time point (dots) are shown with the
use of estimates observed in the blood compartment (A) or
expected for the lymph node compartment (B). The receptor
growth rate p(t) and the disappearance rate d(t) during the course
of the therapy were assumed to follow the observed proportion of
Ki67⫹(proliferating) CD3⫹CD127⫹T cells and an exponential
decay kinetics, respectively (C). The mean values of low- and
high-binding affinities from the 2 donors were used for simula-
tions.
3252 HODGE et al BLOOD, 22 SEPTEMBER 2011 䡠VOLUME 118, NUMBER 12
For personal use only.on December 13, 2016. by guest www.bloodjournal.orgFrom
Correspondence: Irini Sereti, Clinical and Molecular Retrovirol-
ogy Section, Laboratory of Immunoregulation, NIAID, NIH, 10
Center Dr, Bldg 10, Rm 11B07A, Bethesda MD 20892; e-mail:
isereti@niaid.nih.gov.
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IL-7 LEVELS IN TREATED PATIENTS WITH HIV 3253BLOOD, 22 SEPTEMBER 2011 䡠VOLUME 118, NUMBER 12
For personal use only.on December 13, 2016. by guest www.bloodjournal.orgFrom
online July 21, 2011 originally publisheddoi:10.1182/blood-2010-12-323600
2011 118: 3244-3253
JoAnn M. Mican, Chang Paik, Paula DeGrange, Michele Di Mascio and Irini Sereti
Jessica N. Hodge, Sharat Srinivasula, Zonghui Hu, Sarah W. Read, Brian O. Porter, Insook Kim,
suggest a primary mechanism of receptor-mediated clearance
Decreases in IL-7 levels during antiretroviral treatment of HIV infection
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