Enhancement of human antigen-specific memory T-cell responses by interleukin-7 may improve accuracy in diagnosing tuberculosis.
ABSTRACT Children and immunocompromised adults are at an increased risk of tuberculosis (TB), but diagnosis is more challenging. Recently developed gamma interferon (IFN-gamma) release assays provide increased sensitivity and specificity for diagnosis of latent TB, but their use is not FDA approved in immunocompromised or pediatric populations. Both populations have reduced numbers of T cells, which are major producers of IFN-gamma. Interleukin 7 (IL-7), a survival cytokine, stabilizes IFN-gamma message and increases protein production. IL-7 was added to antigen-stimulated lymphocytes to improve IFN-gamma responses as measured by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT) assay. Antigens used were tetanus toxoid (n = 10), p24 (from human immunodeficiency virus [HIV], n = 9), and TB peptides (n = 15). Keyhole limpet hemocyanin was used as a negative control, and phytohemagglutinin was the positive control. IL-7 improved antigen-specific responses to all antigens tested including tetanus toxoid, HIV type 1 p24, and TB peptides (ESAT-6 and CFP-10) with up to a 14-fold increase (mean = 3.8), as measured by ELISA. Increased IFN-gamma responses from controls, HIV-positive patients, and TB patients were statistically significant, with P values of <0.05, 0.01, and 0.05, respectively. ELISPOT assay results confirmed ELISA findings (P values of <0.01, 0.02, and 0.03, respectively), with a strong correlation between the two tests (R(2) = 0.82 to 0.99). Based on average background levels, IL-7 increased detection of IFN-gamma by 39% compared to the level with antigen alone. Increased production of IFN-gamma induced by IL-7 improves sensitivity of ELISA and ELISPOT assays for all antigens tested. Further enhancement of IFN-gamma-based assays might improve TB diagnosis in those populations at highest risk for TB.
- SourceAvailable from: informahealthcare.com[Show abstract] [Hide abstract]
ABSTRACT: The current state-of-the-art tests for infection with Mycobacterium tuberculosis - the IFN-γ release assays - rely on accurate measurement of the cytokine IFN-γ. Many other potential biomarkers are expressed in concert with IFN-γ, and IP-10 in particular has shown promising results. IP-10 is produced in large amounts, allowing for the development of new and simplified test platforms, such as lateral flow. In this review, we summarize the results of 22 clinical studies exploring the use of IP-10 as an alternative marker to IFN-γ. The studies report that diagnostic accuracy of IP-10 is on par with IFN-γ, but also that IP-10 may be more robust in young children and in HIV-infected individuals with low CD4 cell counts. We conclude the review by presenting limitations of the published works and outline recent developments and future directions.Expert Review of Molecular Diagnostics 03/2012; 12(2):175-87. · 4.09 Impact Factor
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ABSTRACT: Patients with end-stage renal disease (ESRD) and latently infected with Mycobacterium tuberculosis (LTBI) are at higher risk to develop tuberculosis (TB) than healthy subjects. Interferon-gamma release assays (IGRAs) were reported to be more sensitive than tuberculin skin tests for the detection of infected individuals in dialysis patients. On 143 dialysis patients prospectively enrolled, we compared the results from the QuantiFERON®-TB Gold assay (QFT), to those of an IGRA in response to in vitro stimulation of circulating mononuclear cells with the mycobacterial latency antigen Heparin-Binding Haemagglutinin purified from Mycobacterium bovis BCG (native HBHA, nHBHA). Seven patients had a past history of active TB and 1 had an undetermined result with both IGRAs. Among the other 135 patients, 94 had concordant results with the QFT and nHBHA-IGRA, 40.0% being negative and therefore not latently infected, and 29.6% being positive and thus LTBI. Discrepant results between these tests were found for 36 patients positive only with the nHBHA-IGRA and 5 only with the QFT. The nHBHA-IGRA is more sensitive than the QFT for the detection of LTBI dialysis patients, and follow-up of the patients will allow us to define the clinical significance of discrepant results between the nHBHA-IGRA and the QFT.PLoS ONE 01/2013; 8(8):e71088. · 3.73 Impact Factor
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ABSTRACT: Background Recent interferon gamma (IFN-γ)-based studies have identified novel Mycobacterium tuberculosis (M.tb) infection phase-dependent antigens as diagnostic candidates. In this study, the levels of 11 host markers other than IFN-γ, were evaluated in whole blood culture supernatants after stimulation with M.tb infection phase-dependent antigens, for the diagnosis of TB disease. Methodology and Principal Findings Five M.tb infection phase-dependent antigens, comprising of three DosR-regulon-encoded proteins (Rv2032, Rv0081, Rv1737c), and two resucitation promoting factors (Rv0867c and Rv2389c), were evaluated in a case-control study with 15 pulmonary TB patients and 15 household contacts that were recruited from a high TB incidence setting in Cape Town, South Africa. After a 7-day whole blood culture, supernatants were harvested and the levels of the host markers evaluated using the Luminex platform. Multiple antigen-specific host markers were identified with promising diagnostic potential. Rv0081-specific levels of IL-12(p40), IP-10, IL-10 and TNF-α were the most promising diagnostic candidates, each ascertaining TB disease with an accuracy of 100%, 95% confidence interval for the area under the receiver operating characteristics plots, (1.0 to 1.0). Conclusions Multiple cytokines other than IFN-γ in whole blood culture supernatants after stimulation with M.tb infection phase-dependent antigens show promise as diagnostic markers for active TB. These preliminary findings should be verified in well-designed diagnostic studies employing short-term culture assays.PLoS ONE 06/2012; 7(6):e38501. · 3.73 Impact Factor
CLINICAL AND VACCINE IMMUNOLOGY, Oct. 2008, p. 1616–1622
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 15, No. 10
Enhancement of Human Antigen-Specific Memory T-Cell Responses
by Interleukin-7 May Improve Accuracy in Diagnosing Tuberculosis?†
Marsha Feske,1,2,3Rodolfo J. Nudelman,2Miguel Medina,2Justin Lew,3Manisha Singh,2
Jacob Couturier,2Edward A. Graviss,3and Dorothy E. Lewis2*
University of Texas School of Public Health1and Departments of Immunology2and Pathology,3
Baylor College of Medicine, Houston, Texas
Received 23 May 2008/Returned for modification 27 June 2008/Accepted 13 August 2008
Children and immunocompromised adults are at an increased risk of tuberculosis (TB), but diagnosis is
more challenging. Recently developed gamma interferon (IFN-?) release assays provide increased sensitivity
and specificity for diagnosis of latent TB, but their use is not FDA approved in immunocompromised or
pediatric populations. Both populations have reduced numbers of T cells, which are major producers of IFN-?.
Interleukin 7 (IL-7), a survival cytokine, stabilizes IFN-? message and increases protein production. IL-7 was
added to antigen-stimulated lymphocytes to improve IFN-? responses as measured by enzyme-linked immu-
nosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT) assay. Antigens used were tetanus
toxoid (n ? 10), p24 (from human immunodeficiency virus [HIV], n ? 9), and TB peptides (n ? 15). Keyhole
limpet hemocyanin was used as a negative control, and phytohemagglutinin was the positive control. IL-7
improved antigen-specific responses to all antigens tested including tetanus toxoid, HIV type 1 p24, and TB
peptides (ESAT-6 and CFP-10) with up to a 14-fold increase (mean ? 3.8), as measured by ELISA. Increased
IFN-? responses from controls, HIV-positive patients, and TB patients were statistically significant, with P
values of <0.05, 0.01, and 0.05, respectively. ELISPOT assay results confirmed ELISA findings (P values of
<0.01, 0.02, and 0.03, respectively), with a strong correlation between the two tests (R2? 0.82 to 0.99). Based
on average background levels, IL-7 increased detection of IFN-? by 39% compared to the level with antigen
alone. Increased production of IFN-? induced by IL-7 improves sensitivity of ELISA and ELISPOT assays for
all antigens tested. Further enhancement of IFN-?-based assays might improve TB diagnosis in those popu-
lations at highest risk for TB.
Gamma interferon (IFN-?) release assays have been used to
detect both latent and active tuberculosis (TB). Compared to
the century-old tuberculin skin test (TST), these assays have
increased specificity and sensitivity for TB diagnosis in select
populations (31–34). IFN-?-based assays are more predictive
than the TST in diagnosing active TB in both human immu-
nodeficiency virus-positive (HIV?) and pediatric populations
(5, 10, 33). However, the FDA has limited the approved use of
IFN-?-based assays for TB diagnosis to adult non-HIV?pop-
ulations. The reduced number of IFN-?-producing T cells in
both HIV?and pediatric populations likely limits the sensitiv-
ity and specificity of these assays (25, 35). Those who are truly
infected with TB may be misclassified as negative (type II
error) and would therefore potentially forego treatment. In-
deed, an 85% drop in the need for treatment for TB was
reported when a combination of TST and QuantiFERON
Gold (QFT-G) testing was performed (47). False-negative re-
sults for TST but not with QFT-G are seen in HIV-infected
adults (33). The inability to confidently diagnose these patients
impacts both TB control and drug-resistant TB prevalence
(43). For HIV-infected persons, the risk for developing TB
doubles within 1 year of infection (43). For children, the risk of
disease after exposure increases up to 80%, especially in those
with HIV infection (30).
Increased diagnostic sensitivity and specificity for TB infec-
tion would improve case management, prevent unnecessary
exposure to strong chemoprophylaxis, impact TB control, and
increase epidemiologic knowledge (28). For these reasons, im-
provement of IFN-? assays has been recognized as a health
priority (21, 30). The main goal of this study was to increase the
amount of IFN-? secreted or the number of effector cells after
antigen-specific stimulation, which might lead to improved sen-
sitivity and specificity of IFN-based assays. The long-term goal
is to improve TB diagnosis in both HIV?and pediatric popu-
Interleukin-7 (IL-7) functions in T-cell development and
homeostasis (11, 12). It stabilizes IFN-? message, enhances cell
viability, and is being studied as a treatment to improve T-cell
responses (3, 4, 24, 27, 37, 39, 44, 48). A recent publication
shows that recombinant human IL-7 preferentially expands
naïve T cells and central memory CD4?T cells, without reg-
ulatory T-cell (T-reg) expansion (44). IL-7 and IL-15 enhance
enzyme-linked immunospot (ELISPOT) assay responses to pu-
rified protein derivative and cytomegalovirus antigens (24). An
additional study used this same combination of cytokines and
measured HIV type 1 (HIV-1)-specific T-cell responses in chil-
dren in the Bronx (New York, NY) (42).
The two most heavily studied IFN-?-based assays for TB
diagnosis are the QFT-G assay (Cellestis) and the T-Spot TB
* Corresponding author. Mailing address: Department of Internal
Medicine, Division of Infectious Diseases, University of Texas Medical
Branch, 301 University Boulevard, Mail Route 0435, Galveston, TX
77555-0567. Phone: (409) 747-0240. Fax: (409) 772-6527. E-mail: dolewis
† Supplemental material for this article may be found at http://cvi
?Published ahead of print on 27 August 2008.
at HOUSTON ACADEMY OF MEDICINE on December 12, 2008
(Oxford Immunotec). The QFT-G uses enzyme-linked immu-
nosorbent assay (ELISA) techniques to quantify the amount of
IFN-? from whole-blood cultures 24 h after stimulation
(Cellestis QuantiFERON-TB Gold package insert; Cellestis,
Valencia, CA). The T-Spot TB uses isolated peripheral blood
mononuclear cells (PBMCs) so that a known quantity of cells
(2.5 ? 105) is used in the assay. The counted cells are incu-
bated with antigen, and the number of antigen-specific cells is
measured 16 to 20 h after stimulation (10). Studies exploring
the correlation between these assays and the TST have yielded
mixed results, likely because of differences in the numbers of
cells used in the assays (2, 8, 10, 26, 34). Studies have not
directly compared the T-Spot and QFT-G assays on identical
counted cell populations with the same antigens.
MATERIALS AND METHODS
Human subjects. HIV?and control subjects were adults (18 years or older)
who had given informed written consent. All work completed on donated blood
was performed under study protocols approved by the Baylor College of Med-
icine Institutional Review Board. Control subjects (n ? 10) had no prior diag-
nosis of TB or HIV. HIV?patients (n ? 9) were either part of an ongoing study
examining long-term HIV?survivors or current patients at a local clinic. All
HIV?subjects had CD4 counts of 200 or greater. All TB patients (TB?) (n ? 15)
were known to be QFT-G positive by the whole-blood method. These were
subjects enrolling in the Houston Tuberculosis Initiative Project (42a). Individual
participants gave informed, written consent prior to submission of clinical spec-
imens, which were then blinded to preserve patient confidentiality. Thus, all TB
patients tested in this study were known to be QFT-G positive.
Optimization of incubation period. Human peripheral blood was obtained
from healthy donors and HIV?donors. PBMCs were isolated by centrifugation
with Histopaque-1077 density gradients. Cells were counted using a hemocytom-
eter, and viability was assessed using trypan blue dye; viability was between 90
and 95%. Cells were cultured in 24-well plates (Corning Incorporated, Corning,
NY) at a concentration of 1 ? 106/ml/well. PBMCs cultured in RPMI plus 10%
fetal calf serum medium alone served as a negative control, and phytohemag-
glutinin (PHA; 2 ?g/ml) served as a positive control. QuantiFERON CMI kits
were used to quantitate the amount of IFN-? in cell culture supernatants.
Dose response of IL-7. Cells from healthy donors were incubated with different
concentrations of IL-7, ranging from 1 ng/ml to 20 ng/ml, to construct titration
curves, with and without PHA at 2 ?g/ml. Activation of the cultured PBMCs was
analyzed by flow cytometry analysis using CD69, CD25, and HLA-DR monoclo-
nal conjugated antibodies, as well as annexin V binding. The ability of IL-7 to
increase the antigen-specific response to different peptides in healthy subjects’
PBMCs and those from HIV-infected patients was assessed by incubation of 1 ?
106cells/ml with tetanus toxoid (1 ?g/ml) and keyhole limpet hemocyanin (KLH;
Postoptimization PBMC isolation and culture. PBMCs were isolated from 2
to 60 ml of lithium heparin-treated whole blood (BD Vacutainer 7068027) using
Histopaque density gradient centrifugation. PBMCs for control and HIV?pop-
ulations were isolated within 8 h of collection. PBMCs for TB?patients were
isolated more than 24 h after collection because a positive QFT-G result was
required to use the sample for testing with our improved assay. Cells were
resuspended in AIM-V medium (Gibco 12055, Invitrogen). Viability of the cells
was examined by trypan blue exclusion (Cellgro MT 25-900-CI; Invitrogen).
PBMCs of all patients were stimulated using the following conditions: no addi-
tion; 10 ng recombinant human IL-7 (R & D Systems); KLH (15) (Calbiochem),
1 ?g/ml for controls and HIV?patients or 8 ?g/ml for TB patients; KLH plus
IL-7 at 10 ng/ml; PHA (Sigma-Aldrich catalog no. 61764) at 1 ?g/ml or 8 ?g/ml;
and the antigens tetanus toxoid (List Biological Laboratories catalog no. 191A)
and HIV-1 p24 (Protein Science Corp. catalog no. 5834) at a concentration of 1
?g/ml or TB peptides ESAT-6 and CFP-10 at 2 ?g/ml each for a final concen-
tration of 8 ?g/ml (Baylor College of Medicine protein core, Houston, TX).
Treated PBMCs were either transferred to 96-well plates (Costar) after stimu-
lation and incubated for 24 h or placed dropwise into ELISPOT assay wells
(Oxford Immunotec) and incubated for 20 h at 37°C and 5% CO2.
Antigen preparation. Corresponding overlapping mixtures of RD1 coded pep-
tides from ESAT-6 and CFP-10 are as effective at eliciting immune responses as
are the more expensive whole-protein antigen counterparts (1, 40). Peptides
from ESAT-6 and CFP-10 were selected for use in testing TB responses (Table
1) and synthesized in the Baylor College of Medicine protein core using ferri-
mannitol ovalbumin chemistry on an ABI433A peptide synthesizer (Applied
Biosystems Inc.). They were purified by high-pressure liquid chromatography
and verified by mass spectrometry and amino acid analysis.
ELISA. QFT-G assay kits (Cellestis) were used for ELISA determinations.
After 24 h of incubation at 37°C, cells were centrifuged and supernatants were
collected. Conjugate (murine anti-human IFN-? horseradish peroxidase) (50 ?l)
was diluted in a mixture of bovine casein, normal mouse serum, and 0.01%
thimerosal and added to precoated plates. Samples and standards were layered
on top and allowed to incubate for 2 h at room temperature. Plates were washed
for a minimum of six cycles. Substrate (hydrogen peroxide and 3,3?,5,5?-tetra-
methylbenzidine) was added to each well, and a 30-min room-temperature in-
cubation followed. After incubation, stop solution (0.5 M H2SO4) was added and
optical densities were read using 450- and 630-nm filters (Dynex MRX II).
Measured optical densities were converted to IU/ml based on the standard curve
specific for each plate.
ELISPOT. T-Spot TB kits (Oxford Immunotec) were used to determine the
number of IFN-?-producing cells by ELISPOT assay. After 20 h of incubation at
37°C, cells were discarded and the plate was washed using 200 ?l phosphate-
buffered saline (PBS) per well. After the plate was washed three times, 50 ?l of
PBS-diluted conjugate (murine anti-human alkaline phosphatase-conjugated
IFN-?) was added. The plate was then incubated at 2 to 8°C for 1 h. The
conjugate was discarded from the plate followed by four washes with PBS.
Substrate (BCIP/NBTplussolution; 50 ?l) was added, and the plate was incubated
at room temperature for 7 min. The plate was washed and allowed to dry. IFN-?
production was indicated by counting spots using an automated reader (CTL
Immunospot S4). Blinded counts were verified using a stereoscope.
Statistical analysis. The data obtained are presented as means and differences
between means and were assessed using the Student t test. Power analysis, using
mean differences from control patients, indicated that a minimum of nine pa-
tients of each group would be necessary to achieve statistical significance. A P
value of ?0.05 was considered significant for HIV?patients (n ? 9), TB?
patients stimulated at 2.5 ? 106(n ? 9), and control patients (n ? 10). In
addition, for all patients, t test findings were verified using Wilcoxon signed-rank
analysis. Using the calculated means, we correlated the ELISPOT assays and
ELISAs for individual patients and report the coefficient of determination (R2).
Dose response of IL-7. Because IL-7 is a survival cytokine for
T cells (24, 37) and is important in homeostasis of human T
cells (11), the addition of IL-7 was used to see whether acti-
vation would be enhanced. We found that CD25 but not CD69
or DR was upregulated after addition of IL-7 to PBMCs from
control volunteers, confirming other results (see Fig. S1a and
S1b in the supplemental material) (6, 41). However, upregu-
lation of CD25 expression occurred only on CD4?and not
CD8?T cells, indicating a specific subpopulation effect when
IL-7 was added without other factors. IL-7 at 2 ng/ml increased
CD25 expression levels (P ? 0.03), and the addition of up to 20
ng/ml had no increased effect on CD25 levels (see Fig. S1c in
the supplemental material). No increase in CD69 or DR ex-
pression was observed after IL-7 addition. No consistent
increase of viability was noted as measured by annexin V
staining. However, IL-7 enhanced the response to PHA as
measured by increased levels of CD25 expression (see Fig. S1c
TABLE 1. Synthetic peptides used in this study
aESAT-6, 6-kDa early secreted antigenic target from Mycobacterium tubercu-
losis; CFP-10, 10-kDa culture filtrate protein from Mycobacterium tuberculosis.
VOL. 15, 2008IL-7 ENHANCES MEMORY T-CELL RESPONSES1617
at HOUSTON ACADEMY OF MEDICINE on December 12, 2008
in the supplemental material). In these cultures, after PHA
stimulation, the CD8?T cells also expressed higher levels of
CD25 after IL-7 addition. IL-7 also increased the amount of
IFN-? produced as detected by ELISA.
Optimization of incubation period. We then tested for an
antigen-specific effect caused by IL-7 addition by using tetanus
toxoid and p24 as antigens. However, 72 h of incubation re-
sulted in elevated background IFN-? production such that
naïve T cells could have been primed (29). IL-7 and dexameth-
asone treatment of cord blood cells causes maturation of T
cells into effector memory cells (46). IL-7 also promotes tran-
sition of T effectors to T memory cells, at least in the mouse
(27). Therefore, we reduced the incubation time to 24 h. Re-
sults show that tetanus toxoid responses and not KLH re-
sponses (nonspecific antigen control) were improved by IL-7
addition (data not shown). Likewise no response above back-
ground to HIV p24 or TB peptides was seen in six controls in
assays using p24 as antigen and four controls in assays using TB
peptides as antigen after 24 h (data not shown). Together,
these data indicate that IL-7 augments antigen-specific T-cell
IFN-? responses as detected by ELISA after 24 h. An in-
creased response to IL-7 alone or de novo induction of naïve
cells apparently can occur after 72 h of exposure to IL-7 (29).
Rationale for use of PMBCs. Although our primary goal in
these studies was to improve IFN-? detection in TB diagnosis,
it was important to examine the robustness of improvement of
IFN-? responses in both healthy individuals and people with
chronic HIV or suspected TB infection. Therefore, we studied
lymphocytic responses to tetanus toxoid, p24, and TB peptides
after IL-7 addition.
We chose to perform these assays with separated PBMCs,
not with whole blood, to quantify and thereby use the same
number of responding cells for all comparisons. Use of PBMCs
also avoided the complication of adding IL-7 to the complex
milieu of whole blood.
Concentration of PBMCs. All patients tested for latent in-
fection by the Houston Tuberculosis Initiative (42a) had a
separate tube of blood set aside for use in this study. To reduce
the cost of testing all suspect TB cases, patient samples were
not used until the QFT-G test was positive (?0.35 IU IFN-?)
which was 24 to 36 h after blood was obtained. PBMCs of
positive patients were then isolated and stimulated. Our initial
procedure of stimulating 250,000 cells in 250 ?l of medium led
to IFN-? production below that recorded 24 h earlier in the
initial QFT-G test. We suspected that the lack of response
(without IL-7 addition) might be because the cells were from
blood that was 24 h old. However, by increasing the cell-to-
volume ratio to increase the concentration of cells, we restored
the amount of IFN-? detected. This increase in cell concen-
tration was done in nine TB patients, using 250,000 PBMCs
resuspended in either 33 ?l, 100 ?l, or 250 ?l to achieve a final
concentration of 7.5 ? 106, 2.5 ? 106, and 1.0 ? 106cells/ml
(see Fig. S2 in the supplemental material), and in one control,
using different numbers of PBMCs at various volumes (see Fig.
S3 in the supplemental material). The data show that, as the
cell volume is decreased, the TB peptide response increases
(see Fig. S2 in the supplemental material). The addition of
IL-7 to 500,000 antigen-stimulated PBMCs suspended in 50 ?l
of medium produced over 17 IU/ml of IFN-? compared to the
amount produced by 500,000 PBMCs in 100 ?l (13.5 IU/ml)
(see Fig. S3 in the supplemental material). Importantly, as the
concentration of PBMCs increased to raise the number of
effector memory cells, the background level of IFN-? mea-
sured in response to IL-7 stimulation remained consistent.
IFN-? detected by ELISA. After optimization of the assay by
reducing the volume, we studied PBMCs from 28 subjects.
These included 10 controls stimulated with tetanus toxoid, nine
subjects with confirmed TB stimulated with TB peptides, and
nine HIV?patients stimulated with HIV-1 p24. The cells were
incubated for 24 h with no stimulation, with antigen (tetanus
toxoid, TB peptides, or HIV-1 p24), with negative-control
KLH antigen, and with positive-control stimulation (PHA).
The amount of IFN-? detected using KLH, with and without
IL-7, remained statistically insignificant compared to that for
unstimulated cultures and cultures stimulated with IL-7 alone
(P, 0.2 and 0.6, respectively), meaning that there was no dif-
ference between the negative control (KLH) and the unstimu-
lated samples after IL-7 addition. However, after stimula-
tion of PBMCs with antigens, up to a 14-fold increase
(mean, 3.8) in IFN-? level was seen after IL-7 supplemen-
tation with statistically significant differences (P ? 0.01) for
all patients. Significant differences (P ? 0.05) were also seen
when comparing the amounts of IFN-? detected from un-
stimulated and negative-control supernatants with and with-
out IL-7 with amounts detected from antigen-stimulated
supernatants (Fig. 1).
The FDA-approved QFT-G test defines a positive test as
0.35 IU/ml or greater. Using the QFT-G reference cutoff to
distinguish positive IFN-? values from negative IFN-? values
for comparison, IL-7 increased detection of IFN-? by 39%
(from 32% [9/28] to 71% [20/28]), despite the 24-h delay in
stimulation for the confirmed QFT-G TB?subjects. Based on
FIG. 1. IL-7 enhances IFN-? production from antigen-stimulated
lymphocytes. Cells were incubated for 24 h with medium, 10 ng IL-7,
KLH (negative-control antigen), KLH plus IL-7, antigen (tetanus tox-
oid, HIV p24, or TB peptides), or antigen plus IL-7 (n ? 28). Super-
natants were then assayed for IFN-? using ELISA. IL-7 enhances
antigen-specific responses 2- to 14-fold in all patients (P ? 0.01), in 9
of 10 control patients (P ? 0.05), in eight of the nine TB patients (P ?
0.05), and in eight of the nine HIV?patients (P ? 0.01). Positive-
control values (PHA, 1.0 ?g/ml) are not shown, as all positive controls
produced IFN-? at levels above background with values ranging from
0.43 to 81.3 IU/ml. Statistically significant differences were also seen
between the IFN-? levels produced in response to antigen and the
levels produced by unstimulated and negative controls (P ? 0.05).
1618FESKE ET AL.CLIN. VACCINE IMMUNOL.
at HOUSTON ACADEMY OF MEDICINE on December 12, 2008
these values, IL-7 addition increased sensitivity by more than
20% for each study population. Increases were evenly distrib-
uted with values as follows: TB, range of 1.26 to 9.2, mean of
3.6, and median of 2.1; HIV, range of 1.0 to 7.3, mean of 3.7,
and median of 2.5; and controls, range of 1.6 to 14.8, mean of
3.2, and median of 3.1 (Fig. 2).
IFN-? effector cells detected by ELISPOT assay. PBMCs
from 18 patients (10 controls, four QFT-G-confirmed latent
TB patients, and four HIV?patients) were incubated for 20 h
under each condition separately (Fig. 3). The number of indi-
vidual T cells secreting IFN-? after negative-control stimula-
tion by KLH, with and without the addition of IL-7, from all
patients remained statistically insignificant compared with the
number of T cells from unstimulated cultures and cultures
stimulated with IL-7 alone. However, in response to antigen up
to a 16-fold increase (mean, 3.4) in ELISPOT assays occurred
after the addition of IL-7 with statistically significant differ-
ences (P ? 0.01) for all patients. Significant differences (P ?
0.05) were also observed when comparing the numbers of
IFN-?-producing cells from unstimulated cell and negative-
control supernatants with and without IL-7 with those from
antigen-stimulated supernatants with and without IL-7. Raw
data from four subjects in each group are shown in Table S1 in
the supplemental material. Overall the ELISPOT assay results
mirrored and confirmed the QFT-G findings.
ELISPOT/ELISA correlation. We isolated PBMCs and then
used stimulating conditions previously described prior to per-
forming the ELISA and ELISPOT assays. We found that
ELISA detection of IFN-? correlated well with the number of
FIG. 2. Addition of IL-7 increases ELISA sensitivity. The FDA-approved QFT-G defines a positive result as 0.35 IU/ml or greater. This graph
displays the increased sensitivity in response to antigen stimulation after addition of IL-7 in all study subjects (10 controls, nine TB patients, and
nine HIV?patients). Increases with the addition of IL-7 were evenly distributed in each study group: TB response to TB peptides (range ? 1.26
to 9.2, mean ? 3.6, median ? 2.1), HIV response to HIV-1 p24 (range ? 1.0 to 7.3, mean ? 3.5, median ? 2.8), and control response to tetanus
toxoid (range ? 1.6 to 14.8, mean ? 3.2, median ? 3.1). When the QFT-G value of 0.35 IU/ml is used, IL-7 increased sensitivity, from 30% (7/23)
to 70% (16/23).
FIG. 3. Increase in effector cell number with IL-7 addition. Cells
were incubated in ELISPOT plates for 20 h with medium alone, 10 ng
IL-7, KLH (negative-control antigen), KLH plus IL-7, antigen (tetanus
toxoid [TT], HIV p24, or TB peptides (ESAT-6 and CFP-10), or
antigen plus IL-7 (n ? 18). Plates were developed, and spots were
counted using an automated reader. IL-7 enhances antigen-specific
responses up to 16-fold (mean ? 3) in all patients (P ? 0.01), in all
control patients (P ? 0.01), in all TB patients (P ? 0.03), and in all of
the HIV?patients (P ? 0.02). Statistically significant differences were
also seen between the IFN-? levels produced in response to antigen
and the levels produced by unstimulated and negative controls (P ?
VOL. 15, 2008 IL-7 ENHANCES MEMORY T-CELL RESPONSES1619
at HOUSTON ACADEMY OF MEDICINE on December 12, 2008
spots detected by ELISPOT assay (Fig. 4). For control patients
the ELISA and ELISPOT results gave similar individual coef-
ficients of determination with a range of 0.92 to 1.0 (? ? 0.98,
? ? 0.04). For HIV?patients and TB patients, the range of
individual coefficients of determination was 0.83 to 0.99 (? ?
0.92, ? ? 0.08) and 0.93 to 0.99 (? ? 0.98, ? ? 0.02), respec-
tively. The mean coefficient for all patients tested using both
diagnostic techniques was R2? 0.96 with a standard deviation
In this study, we measured the effects of IL-7 on IFN-?
production in cells from three different groups of subjects. IL-7
enhanced antigen-specific responses 2- to 14-fold in all patients
(P ? 0.01), in 9 of 10 control patients with tetanus toxoid
stimulation (P ? 0.05), in eight of the nine TB patients (P ?
0.05), and in eight of the nine HIV?patients (P ? 0.01) (n ?
9) (Fig. 2). IL-7 did not enhance non-antigen-specific re-
sponses as shown by the use of IL-7 alone and the negative
control, KLH. Our results confirm and add to the findings of
Jennes et al. (22). Using the QFT-G value of 0.35 IU/ml de-
fined as a positive test, for comparative purposes, more than a
20% increase in the amount of IFN-? was found for each
population studied (controls and HIV?and TB?patients)
with an overall improvement of 39%.
The increased IFN-? production in both healthy controls
and diseased people is an important finding because other
factors can impair IFN-? production and confound responses
in both TB and HIV?patients (17–19). The decreased ability
to produce detectable levels of IFN-? in people with TB has
been attributed to the effects of increased levels of T-regs (14,
38) and transforming growth factor ? (11, 18) and could also be
due to increased cellular apoptosis (16). Spontaneous and an-
tigen-induced programmed cell death levels are elevated in
patients with TB compared to control subjects (16, 17). T-regs
are increased threefold during active TB infection and can
reduce IFN-? responses (33, 38). In HIV-infected people, nat-
urally occurring CD4?CD25?T-regs suppress both CD4?and
CD8?cell responses (23). In addition, highly active antiretro-
viral therapy suppresses viral loads to stabilize or increase the
number of CD4?cells and, in turn, actually decreases respon-
siveness to HIV antigens (36). Some of these mechanisms,
such as a decrease in T-regs, are reduced by IL-7 (20, 39) and
may be a further explanation for our finding of increased
production of IFN-? upon IL-7 addition. However, a key factor
is that numbers of T memory cells are reduced in HIV infec-
Higher levels of IFN-? were detected from the same number
of PBMCs if the volume was reduced. This was first noticed in
our TB population (see Fig. S2 in the supplemental material)
and confirmed with tetanus toxoid stimulation in a control (see
Fig. S3 in the supplemental material). The response to IL-7
addition alone, however, remained unchanged. By increasing
the cell-to-volume ratio and theoretically cell-to-cell interac-
tions, we increased the amount of IFN-? without increasing the
background level of IFN-?. We propose that by decreasing the
volume, thereby crowding the cells, the probability of receptor-
ligand interactions and potentially the sampling of antigen by
dendritic cells were increased. Further studies are in progress
to determine key players in the upregulation of IFN-? produc-
tion caused by IL-7 addition.
As possible evidence that IFN-? production by antigen-spe-
cific cells may provide a method for detection of the response
to infection, a previous study compared amounts of IFN-? in
response to purified protein derivative, ESAT-6, and PHA,
using ELISA, ELISPOT, and reverse transcription-PCR tech-
niques. This study reported that antigen stimulation with
ESAT-6 peptide increased IFN-? mRNA levels exponentially,
but similar increases in IFN-? protein production were not
found (9). This indicates that inhibition in translation may be
present. In light of this study and the known ability of IL-7 to
stabilize IFN-? message, we suggest that IL-7 allows more
protein to be processed from the induced or existing message,
an idea which will be tested in future studies. By using IL-7 to
increase and stabilize IFN-? production, latently infected in-
dividuals with few or impaired effector cells have an increased
probability of being detected as TB reactive.
To examine whether increased IFN-? production resulted in
an increase in the number of effector cells, we used ELISPOT
assays (Fig. 3). Our findings of increased effector cell numbers
after addition of IL-7 are in agreement with previous results
from cryopreserved specimens (22).
Since the development of IFN-?-based assays, many studies
have compared their effectiveness to that of the TST. Findings
of improved sensitivity vary widely, and the most recent study
showed concern about the negative predictive value of IFN-?-
FIG. 4. Correlations between ELISA and ELISPOT results for two
tetanus toxoid-stimulated control patients (A and B), two TB?pep-
tide-stimulated patients (C and D), and two HIV?HIV-1 p24-stimu-
lated patients (E and F). Correlations include ELISA and ELISPOT
results for all conditions and are specific to the individual patients.
Coefficient of determination values for graphs shown are two examples
from each population sampled: controls (A [R2? 0.99] and B [R2?
0.99]), tetanus toxoid patients (C [R2? 0.99] and D [R2? 0.99]), and
HIV?patients (E [R2? 0.99] and F [R2? 0.99]).
1620 FESKE ET AL.CLIN. VACCINE IMMUNOL.
at HOUSTON ACADEMY OF MEDICINE on December 12, 2008
based assays compared to that of TST (7). The QFT-G assay
tests for TB by stimulating 1 ml of whole blood, and the
ELISPOT assay relies on isolation of PBMCs prior to antigen
stimulation. When we tested with the same cells and antigens,
we found that results from ELISA and ELISPOT assays cor-
related well (Fig. 4). The mean correlation coefficient (R2) for
all patients tested using both diagnostic techniques was 0.96.
This correlation is higher than that previously reported (13)
and indicates that IL-7 increases the amount of IFN-? pro-
duced by increasing the number of responding effector cells
and that QFT-G and ELISPOT tests detect IFN-? with nearly
equal sensitivities. Other studies comparing these two assays
have been compromised by dissimilar incubation times and
stimulation conditions (9, 31). The failure to count the number
of cells and adjust the concentration used is also an inherent
limitation of the whole-blood assay, especially in cases where
CD4 numbers are reduced, as in HIV infection.
A current limitation of IFN-?-based assays is the require-
ment of sample processing within 12 h of collection (Cellestis
QuantiFERON-TB Gold package insert). This is especially
difficult in resource-poor countries. Indeed, discrepancies from
two different sites in Africa correlated with the time that it took
to process the samples (9). We show that, after IL-7 addition,
TB-specific IFN-? can still be detected despite a greater-than-
24-h period between blood collection and PBMC isolation.
This may be due to message stabilization by IL-7 and/or en-
hancement of viability, which we will examine in future studies.
In conclusion, IL-7 increases IFN-? detection in response to
antigen, which has important public health implications. Im-
provement of IFN-? diagnostic sensitivity might result in im-
provement in the detection of latent infection, the treatment of
pediatric and HIV-infected individuals, and strengthened TB
control. Further studies will investigate if IL-7 acts similarly in
pediatric TB patients.
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