# Lack of Disease Specificity Limits the Usefulness of In Vitro Costimulation in HIV- and HCV-Infected Patients

Authors: Stefanie Kuerten; Tobias R. Schlingmann; Tarvo Rajasalu; Doychin N. Angelov; Paul V. Lehmann; Magdalena Tary-Lehmann
Journal: Clinical and Developmental Immunology
 (2008)

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## Abstract

Measurements of antigen-specific T cell responses in chronic diseases are limited by low frequencies of antigen-specific cells in the peripheral blood.  Therefore, attempts have been made to add costimulatory molecules such as anti-CD28 or IL-7/IL-15 to ELISPOT assays to increase sensitivity.  While this approach has been successful under certain circumstances, results are often inconsistent.  To date, there are no comprehensive studies directly comparing the in vitro effects of multiple costimulatory molecules in different disease settings.  Therefore, in the present study we tested the effects of IL-7/IL-15, IFN-α, anti-ICOS, and anti-CD28 on antigen-specific T cell responses in patients infected with HCV or HIV versus healthy individuals. Our data show that none of the aforementioned molecules could significantly increase ELISPOT sensitivity, neither in HCV nor in HIV.  Moreover, all of them caused false-positive responses to HCV and HIV antigens in healthy individuals. Our results question the broad use of in vitro costimulation.


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## Body

## 1. Introduction

The measurement
of immune responses to disease-specific antigens has become increasingly
important in chronic viral infections. Particularly T cell responses have proven to be a valuable tool both for
vaccine development and immune monitoring [1, 2]. Examining the phenotype and activation status
of T cells (e.g., by FACS analysis) or their cytokine secretion profile (e.g.,
in ELISPOT assays) not only provides helpful information on the immunogenicity
of individual viral antigens, but also reflects the patient's current state of
immunocompetence, a vital piece of information for treatment and prognosis.One
major obstacle, however, is the intrinsically low-frequency and cytokine
productivity of T cells in hosts suffering from chronic disease. Not only infections like HIV that directly
impair T cell function, but also viral infections that target cells outside the
immune system, such as HCV, have been shown to decrease T cell reactivity to
viral antigensin vitro [3–10]. As shown by Yonkers
et al., this effect is even more pronounced in cases of HIV/HCV coinfection
[11]. This alteration of T cell function
by chronic viral infections makes it almost impossible to accurately quantify
the disease-specific T cell repertoire in
vitro.A great deal of hope has therefore been placed into the use of in vitro costimulation. Adding molecules such as agonistic anti-CD28
antibodies or cytokines like IL-7 and IL-15 to FACS and ELISPOT assays is
intended to optimize the interaction between T cells and APC [11–13]. The rationale of a strong second signal
compensating for a weak first signal and/or a high T cell activation threshold
is indeed appealing. Pathogen-specific T
cells would no longer escape detection due to suboptimal in vitro stimulation, but would be reliably activated above
threshold and thus become detectable. Following this rationale, in
vitro stimulation would allow for a precise quantification of each
individual's antigen-specific T cell repertoire.Several
studies have indeed shown increased T cell responses after in vitro costimulation; Jennes et al. demonstrated that the presence
of IL-7 and IL-15 during T cell stimulation in ELISPOT assays significantly
increases the frequency of PPD-specific and CMV-specific T cells [12]. Calarota et al. later demonstrated that IL-15
alone suffices to enhance IFN-γ production to both simian-human
immunodeficiency virus and HIV antigens in infected macaques [14]. Finally, Ott et al. demonstrated that
agonistic anti-CD28 antibody increases the IFN-γ response to tetanus toxoid,
mumps antigen, CMV, and EBV peptides in healthy individuals as well as
responses to NS3 protein in HCV patients and proinsulin along with islet cell
antigen in type 1 diabetes patients [13].Other
studies, however, indicate a rather limited effect of in vitro costimulation. As Yonkers et al. demonstrated, memory-effector CD8 cells show reduced
responsiveness to CD28 costimulation in HCV/HIV coinfection [11]. Subudhi et al. carefully delineated the
opposing effects of B7 family members on immune responses, and showed that
immune modulators frequently lead to coinhibition rather than the desired
stimulation [15].Much
uncertainty therefore remains mainly because no human study to date has
systematically compared multiple types of in vitro costimulation in the setting of different viral
infections. Moreover, most studies
compare stimulated versus unstimulated responses within infected patients. Little attention, however, has so far been paid
to the effect of in vitro
costimulation in healthy, uninfected individuals. How specific are the responses obtained after
costimulation? Is the desired increase
in assay sensitivity compromised by decreased specificity?Our
present study sheds some light upon these essential questions.  We systematically compared the effects of four
different methods of in vitro
costimulation: IL-7/IL-15, IFN-α,
anti-ICOS, and anti-CD28. Using IFN-γ
ELISPOT assays with and without costimulation, we measured the T cell response
to disease-specific antigens in 10 HCV patients, 10 HIV patients, and 6 healthy
individuals. Our results lead to three
major conclusions. Firstly, in HCV
patients, significant increases in T cell reactivity could only be seen with
one particular antigen (NS3 protein) when using either IL-7/IL-15 or
anti-CD28. Secondly, in HIV patients,
none of the four types of in vitro
costimulation led to a statistically significant response increase, regardless
of the antigen used. Lastly and most
importantly, all four methods of costimulation led to false-positive responses
to HCV and HIV antigens in healthy individuals.


## 2. Materials and Methods

### 2.1. Subjects and Sample Collection

Peripheral
blood was obtained from 10 HIV positive and 10 HCV-infected subjects—the former from
the Special Immunology Unit at University Hospitals of Cleveland,
and the latter from the Cleveland Veterans Affairs Medical Center and University Hospitals of Cleveland.
HIV infection was defined as a positive result for ELISA or other licensed HIV
antibody tests, as well as previously detectable plasma HIV RNA. HCV infection was defined as detectable serum
HCV antibodies and RNA. The six healthy controls tested were members of our and
adjoining laboratories, and were negative for both HIV and HCV antibodies. For all participants, PBMCs were isolated
from 40–100 mL of
heparinized blood by standard Ficoll density-gradient centrifugation (IsoPrep,
Robbins Scientific Corp., Sunnyvale,
Calif, USA)
and immediately used for ELISPOT analysis. All studies were performed under the approval of the Institutional
Review Board for Human Investigation at the University Hospitals of Cleveland and the Cleveland Veterans Affairs Medical Center.


### 2.2. ELISPOT Assays and Image Analysis

ImmunoSpot plates (Cellular Technology Ltd., Cleveland, Ohio, USA) were coated with IFN-γ capture antibody mAb M700A (Endogen, Woburn, Mass, USA) in PBS (3μg/mL) and placed at
4°C overnight. The plates were then blocked with PBS
containing 1% BSA (Sigma-Aldrich, St.
Louis, Mo, USA) for 1 hour and washed three
times with PBS.  Freshly isolated PBMCs
were plated in RPMI-1640 (BioWhittaker, Walkersville,Md, USA)
supplemented with 100 U/mL penicillin, 100μg/mL streptomycin, 2 mM L-glutamine, and
10% pooled heat-inactivated human AB serum. For all experiments, 3×105 PBMCs were plated per well in the
presence or absence of IL-7/IL-15 (final concentration of 5 ng/mL; Cell
Sciences, Canton, Mass, USA), IFN-α (final concentration of 1000 U/mL;
Biosource, Camarillo, Calif, USA), agonistic anti-ICOS antibody (final
concentration of 1μg/mL; eBioscience, San Diego, Calif, USA),
or agonistic anti-CD28 antibody (final concentration of 1μg/mL; BD Pharmingen, San Jose, Calif,
USA). The following HIV antigen pools
were used: Pool 1 (comprised of HIV-1
peptides A0301 pol, A201 pol 2, A24 nef8, and A201 nef1),
Pool 2 (comprised of HIV-1 peptides A24 gp41, B27 gp120, A301 p17, and A201 nef2), and Pool 3 (comprised of HIV-1
peptides B27 gp41, A201 nef3, B51 nef, and A24 gp120). All peptide pools were used at 10μg/mL. All HIV peptides were obtained from the NIH AIDS Research and Reference
Reagent Program, Division of AIDS, NIAID, NIH. Recombinant HCV core NS3 protein was supplied by Chiron (Emeryville, Calif,
USA) and used
at a concentration of 10μg/mL. A set of 73 MHC class I-restricted HCV peptides representing portions
of the HCV genotype 1 protein sequence, based upon previously
described CD8 determinants [16–19], were synthesized by the multipin
technique (Chiron, Emeryville, Calif, USA.) and pooled together as a set
(Pepset 4, used at 3.4μM). Phytohemagglutinin (PHA) was used as positive control in all assays, and
was obtained from Sigma-Aldrich (10μg/mL). Negative control wells contained PBMC with a medium in the presence or
absence of the costimulatory agents as mentioned above. After 24 hours of incubation at 37°C and 7%
CO2, the plates were washed with PBS and PBS/TWEEN, and IFN-γ biotinylated detection antibody mAB M701 (Endogen, 2μg/mL) was added. The antibody was diluted in PBS containing 1%
BSA and 0.025% TWEEN (Fisher Scientific International Inc., Hampton, NH, USA). The plates were incubated at 4°C
overnight. Then they were washed
three times with PBS/TWEEN, and subsequently streptavidin-HRP conjugate
(DAKO, Carpinteria, Calif, USA)
was added at 1/2000 dilution, incubated for 2 hours at room
temperature, and removed by washing twice with PBS and
PBS/TWEEN. The spots were visualized by
adding HRP substrate 3-amino-9-ethylcarbozole (Pierce, Rockford, Ill,
USA). The reaction was stopped by rinsing the
plate with distilled water when distinct spots were visible
macroscopically. Plates were dried
overnight and images of the ELISPOT wells were captured with an
ImmunoSpot Series 5 Analyzer (Cellular Technology Ltd.). Image
analysis of the ELISPOT results was performed with the ImmunoSpot 5
Analysis Software (Cellular Technology Ltd.).


### 2.3. Statistical Analysis

Mann-Whitney rank
sum test was calculated using SigmaStat (Version 7; SPSS Inc., San Jose, Calif,
USA) to test for significant differences between values obtained with and
without the addition of IL-7/IL-15, IFN-α, anti-ICOS, or anti-CD28,
respectively. A probability value of P  ≤  .05 was considered to be statistically significant.


## 2.1. Subjects and Sample Collection

Peripheral
blood was obtained from 10 HIV positive and 10 HCV-infected subjects—the former from
the Special Immunology Unit at University Hospitals of Cleveland,
and the latter from the Cleveland Veterans Affairs Medical Center and University Hospitals of Cleveland.
HIV infection was defined as a positive result for ELISA or other licensed HIV
antibody tests, as well as previously detectable plasma HIV RNA. HCV infection was defined as detectable serum
HCV antibodies and RNA. The six healthy controls tested were members of our and
adjoining laboratories, and were negative for both HIV and HCV antibodies. For all participants, PBMCs were isolated
from 40–100 mL of
heparinized blood by standard Ficoll density-gradient centrifugation (IsoPrep,
Robbins Scientific Corp., Sunnyvale,
Calif, USA)
and immediately used for ELISPOT analysis. All studies were performed under the approval of the Institutional
Review Board for Human Investigation at the University Hospitals of Cleveland and the Cleveland Veterans Affairs Medical Center.


## 2.2. ELISPOT Assays and Image Analysis

ImmunoSpot plates (Cellular Technology Ltd., Cleveland, Ohio, USA) were coated with IFN-γ capture antibody mAb M700A (Endogen, Woburn, Mass, USA) in PBS (3μg/mL) and placed at
4°C overnight. The plates were then blocked with PBS
containing 1% BSA (Sigma-Aldrich, St.
Louis, Mo, USA) for 1 hour and washed three
times with PBS.  Freshly isolated PBMCs
were plated in RPMI-1640 (BioWhittaker, Walkersville,Md, USA)
supplemented with 100 U/mL penicillin, 100μg/mL streptomycin, 2 mM L-glutamine, and
10% pooled heat-inactivated human AB serum. For all experiments, 3×105 PBMCs were plated per well in the
presence or absence of IL-7/IL-15 (final concentration of 5 ng/mL; Cell
Sciences, Canton, Mass, USA), IFN-α (final concentration of 1000 U/mL;
Biosource, Camarillo, Calif, USA), agonistic anti-ICOS antibody (final
concentration of 1μg/mL; eBioscience, San Diego, Calif, USA),
or agonistic anti-CD28 antibody (final concentration of 1μg/mL; BD Pharmingen, San Jose, Calif,
USA). The following HIV antigen pools
were used: Pool 1 (comprised of HIV-1
peptides A0301 pol, A201 pol 2, A24 nef8, and A201 nef1),
Pool 2 (comprised of HIV-1 peptides A24 gp41, B27 gp120, A301 p17, and A201 nef2), and Pool 3 (comprised of HIV-1
peptides B27 gp41, A201 nef3, B51 nef, and A24 gp120). All peptide pools were used at 10μg/mL. All HIV peptides were obtained from the NIH AIDS Research and Reference
Reagent Program, Division of AIDS, NIAID, NIH. Recombinant HCV core NS3 protein was supplied by Chiron (Emeryville, Calif,
USA) and used
at a concentration of 10μg/mL. A set of 73 MHC class I-restricted HCV peptides representing portions
of the HCV genotype 1 protein sequence, based upon previously
described CD8 determinants [16–19], were synthesized by the multipin
technique (Chiron, Emeryville, Calif, USA.) and pooled together as a set
(Pepset 4, used at 3.4μM). Phytohemagglutinin (PHA) was used as positive control in all assays, and
was obtained from Sigma-Aldrich (10μg/mL). Negative control wells contained PBMC with a medium in the presence or
absence of the costimulatory agents as mentioned above. After 24 hours of incubation at 37°C and 7%
CO2, the plates were washed with PBS and PBS/TWEEN, and IFN-γ biotinylated detection antibody mAB M701 (Endogen, 2μg/mL) was added. The antibody was diluted in PBS containing 1%
BSA and 0.025% TWEEN (Fisher Scientific International Inc., Hampton, NH, USA). The plates were incubated at 4°C
overnight. Then they were washed
three times with PBS/TWEEN, and subsequently streptavidin-HRP conjugate
(DAKO, Carpinteria, Calif, USA)
was added at 1/2000 dilution, incubated for 2 hours at room
temperature, and removed by washing twice with PBS and
PBS/TWEEN. The spots were visualized by
adding HRP substrate 3-amino-9-ethylcarbozole (Pierce, Rockford, Ill,
USA). The reaction was stopped by rinsing the
plate with distilled water when distinct spots were visible
macroscopically. Plates were dried
overnight and images of the ELISPOT wells were captured with an
ImmunoSpot Series 5 Analyzer (Cellular Technology Ltd.). Image
analysis of the ELISPOT results was performed with the ImmunoSpot 5
Analysis Software (Cellular Technology Ltd.).


## 2.3. Statistical Analysis

Mann-Whitney rank
sum test was calculated using SigmaStat (Version 7; SPSS Inc., San Jose, Calif,
USA) to test for significant differences between values obtained with and
without the addition of IL-7/IL-15, IFN-α, anti-ICOS, or anti-CD28,
respectively. A probability value of P  ≤  .05 was considered to be statistically significant.


## 3. Results

### 3.1. Characteristics of the Study Subjects

The clinical
characteristics of the subjects evaluated for HCV- and HIV-specific T cell functions as well as of healthy individuals are
listed in Table1. Age and gender are
listed for all subjects. For HCV-infected patients, HCV genotype, plasma HCV
RNA levels, and platelet counts are given. Liver function is represented by serum albumin levels, ALT levels, and
total bilirubin levels. For HIV-infected
patients, CD4 cell counts and plasma HIV RNA levels are listed. The ratio of treated to untreated patients is
given for both patient groups.Table 1
Clinical characteristics of study subjects.
Data are given as means  ±  SD, unless otherwise specified. HCV: hepatitis C virus; HIV: human
immunodeficiency virus; ALT: alanine aminotransferase; N/A: not applicable.
HCVHIVHealthyGendern=10 malesn=4 malesn=4 malesn=6 femalesn=2 femalesAge52.75±2.334.8±5.929.6±3.4CD4 cell count,106 cells/LN/A451.3±134.9N/APlasma HIV RNA level,
IU/mL0.0±0.08,685.1±15,122.80.0±0.0HCV genotypeType 1:n=9N/AN/AType 2:n=1Plasma HCV RNA level,
IU/mL1,699,275±1,505,1210.0±0.00.0±0.0Albumin, g/dL3.9±0.5N/AN/AALT, IU/L80.3±67.2N/AN/ATotal bilirubin level,
mg/dL0.5±0.3N/AN/APlatelet count,103 cells/mm3211.6±55.8N/AN/AOn treatment4/108/100/10


### 3.2. Increased Responses to NS3 Protein in HCV-Infected Patients Only after In Vitro
Costimulation with IL-7/IL-15 or Anti-CD28

In order to
compare the effects of different types of in vitro costimulation in 10 HCV-infected patients, we tested
recall responses to NS3 protein and Pepset 4 peptide pool in IFN-γ ELISPOT
assays. Results are shown in Figure 1.Increased responses to NS3 protein in
HCV-infected patients only after in vitro costimulation with IL-7/IL-15 or
anti-CD28. PBMCs of 10 HCV-infected
patients were tested in IFN-γ ELISPOT assays for their recall responses to NS3
protein and Pepset 4 peptide pool in the absence (a) or presence of IL-7/IL-15
(b), IFN-α (c), anti-ICOS (d), or CD28 (e). For each individual, the mean spot count of duplicate wells is
shown. Horizontal bars indicate the
means of all individual responses to the respective antigens. P values are given for means that are
significantly different from baseline values in (a). Symbols in (a) also apply to (b)–(e).
(a)(b)(c)(d)(e)Figure1(a) illustrates the low
baseline responses to NS3 protein and Pepset 4 pool (2.5±2.5 and 6.8±9.3
spots above medium, resp.) as frequently described in the literature in
the case of chronic infection [3–10]. The presence of IL-7/IL-15 during in
vitro T cell stimulation led to a significant ten-fold upregulation of
IFN-γ recall responses to NS3 protein (Figure 1(b); P  =  .02). Three of the patients also showed increased
responses to Pepset 4. However, due to
the wide range of spot numbers, the mean increase was not statistically
significant. Neither IFN-α  (Figure 1(c))
nor anti-ICOS (Figure 1(d)) had a general effect on HCV-specific
responses. Only HCV patients 1 and 7
showed an increased NS3 response upon IFN-α stimulation (P  =  .002 and P  =  .026, resp.). Costimulation with anti-CD28 (Figure 1(e)) showed
similar results compared to IL-7/IL-15 with a significant four-fold increase of
spot numbers (P  =  .021) after recall with NS3 protein. This is consistent with observations made by
Yonkers et al. and Ott et al. showing that HCV-specific T cells remain
responsive to CD28 stimulation during chronic infection [11, 13]. This consistency of results despite
significantly different patient age distributions in the aforementioned studies
also shows that patient age does not seem to influence the effects of in vitro costimulation.


### 3.3. In Vitro Costimulation Has No Effect on Antigen-Specific T Cell Responses in
HIV-Infected Patients

In order to
explore whether the aforementioned types of costimulation have a similar effect
in the case of HIV infection, we tested recall responses to three different HIV
peptide pools in 10 HIV-infected patients. Results are shown in Figure2.In vitro costimulation has no effect on
antigen-specific T cell responses in HIV-infected patients. PBMCs of 10 HIV-infected patients were tested in IFN-γ ELISPOT assays for their recall responses to HIV peptide pools 1, 2,
and 3 (as described in Section 2)
in the absence (a) or presence of IL-7/IL-15 (b), IFN-α (c), anti-ICOS (d), or
CD28 (e). For each individual, the mean
spot count of duplicate wells is shown. Horizontal bars indicate the means of all individual responses to the
respective antigens. Symbols in (a) also
apply to (b)–(e).
(a)(b)(c)(d)(e)Unlike in HCV, some of the patients
showed positive baseline responses to HIV peptides (Figure2(a)). However, none of the costimulatory reagents
could significantly enhance the overall response pattern, as shown in Figures 2(b)–2(e).  None of the mean responses to any of the
antigens was significantly elevated, regardless of the
(interindividually highly variable) baseline reactivity. Results were also identical in both
antiretrovirally treated and untreated patients, showing that antiretroviral
therapy did not seem to influence the effects of in vitro costimulation. This
decreased responsiveness of HIV-infected lymphocytes to costimulation has
previously been described by Borthwick et al. [20] and Trimble et al. [21], and
is attributed to the downregulation of costimulatory molecules such as CD28 on
T cells in the course of chronic HIV infection [22–24].  The precise mechanisms of this defect,
however, are still not understood. Our
data indicate that this effect may not be limited to CD28.


### 3.4. In Vitro Costimulation Causes False-Positive T Cell Responses to HCV and HIV
Antigens in Healthy Individuals

To date, there are
no conclusive data available regarding the effect of in vitro costimulation on HCV- and HIV-specific recall responses
in healthy individuals. This information
is crucial, though, in order to be able to properly interpret positive test
results obtained from assays using in
vitro costimulation. Therefore,
we specifically tested the same HCV and HIV antigens as above in 6 healthy
individuals (Figure3). If in vitro costimulation enhances
existing responses without altering disease specificity, it should not induce
positive responses in healthy individuals.In
vitro costimulation causes false-positive T cell responses to HCV and HIV
antigens in healthy individuals. PBMCs of 6 healthy individuals were
tested in IFN-γ ELISPOT assays for their recall responses to NS3 protein,
Pepset 4 peptide pool, and HIV peptide pools 1, 2, and 3 in the absence (a) or
presence of IL-7/IL-15 (b), IFN-α (c), anti-ICOS (d), or CD28 (e). For each individual, the mean spot count of
duplicate wells is shown. Horizontal
bars indicate the means of all individual responses to the respective
antigens. P values are given for
means that are significantly different from baseline values in (a). Symbols in (a) also apply to (b)–(e).
(a)(b)(c)(d)(e)As shown in Figure3, all four
methods of in vitro
costimulation (Figures 3(b)–3(e)) caused
marked responses to HCV and/or HIV antigens in several healthy
individuals. IL-7/IL-15 (Figure 3(b))
led to a strong positive response to Pepset 4 peptide pool (twenty-fold
increase above baseline; P  =  .026) as well as to HIV pool 1 (ten-fold
increase above baseline; P  =  .030). The mean frequency of NS3 reactive T cells was increased six-fold. Although this shift in mean frequency is not
statistically significant due to the broad range of anti-NS3 responses, the
changes for individual subjects are highly significant (P  =  .002 for
Healthy Control 1; P  =  .005 for Healthy Control 2).Similarly,
IFN-α also induced false-positive responses to NS3 (Figure 3(c)), as seen in
two individuals (P  =  .028 for Healthy Control 1; P  =  .021 for
Healthy Control 2). Interestingly, these
individuals had also displayed susceptibility to IL-7/IL-15 stimulation (Figure
3(b)). In vitro costimulation
with anti-ICOS agonist (Figure 3(d)) upregulated anti-NS3 responses to a lesser
degree. Healthy Control 4 responded with ~300 spots above baseline (P  <  .001). Remarkably, this individual had previously proven nonreactive to
both IL-7/IL-15 and IFN-α. The mean
frequency of Pepset 4 reactive T cells increased four-fold (P  =  .011)
upon ICOS stimulation.Finally,
as shown in Figure3(e), CD28 costimulation also caused false-positive test
results. Healthy Control 4 responded to
NS3 approximately three-fold above baseline (P  =  .006). Moreover, mean
spot counts for Pepset 4 were also increased significantly (P  =  .033).


## 3.1. Characteristics of the Study Subjects

The clinical
characteristics of the subjects evaluated for HCV- and HIV-specific T cell functions as well as of healthy individuals are
listed in Table1. Age and gender are
listed for all subjects. For HCV-infected patients, HCV genotype, plasma HCV
RNA levels, and platelet counts are given. Liver function is represented by serum albumin levels, ALT levels, and
total bilirubin levels. For HIV-infected
patients, CD4 cell counts and plasma HIV RNA levels are listed. The ratio of treated to untreated patients is
given for both patient groups.Table 1
Clinical characteristics of study subjects.
Data are given as means  ±  SD, unless otherwise specified. HCV: hepatitis C virus; HIV: human
immunodeficiency virus; ALT: alanine aminotransferase; N/A: not applicable.
HCVHIVHealthyGendern=10 malesn=4 malesn=4 malesn=6 femalesn=2 femalesAge52.75±2.334.8±5.929.6±3.4CD4 cell count,106 cells/LN/A451.3±134.9N/APlasma HIV RNA level,
IU/mL0.0±0.08,685.1±15,122.80.0±0.0HCV genotypeType 1:n=9N/AN/AType 2:n=1Plasma HCV RNA level,
IU/mL1,699,275±1,505,1210.0±0.00.0±0.0Albumin, g/dL3.9±0.5N/AN/AALT, IU/L80.3±67.2N/AN/ATotal bilirubin level,
mg/dL0.5±0.3N/AN/APlatelet count,103 cells/mm3211.6±55.8N/AN/AOn treatment4/108/100/10


## 3.2. Increased Responses to NS3 Protein in HCV-Infected Patients Only after In Vitro
Costimulation with IL-7/IL-15 or Anti-CD28

In order to
compare the effects of different types of in vitro costimulation in 10 HCV-infected patients, we tested
recall responses to NS3 protein and Pepset 4 peptide pool in IFN-γ ELISPOT
assays. Results are shown in Figure 1.Increased responses to NS3 protein in
HCV-infected patients only after in vitro costimulation with IL-7/IL-15 or
anti-CD28. PBMCs of 10 HCV-infected
patients were tested in IFN-γ ELISPOT assays for their recall responses to NS3
protein and Pepset 4 peptide pool in the absence (a) or presence of IL-7/IL-15
(b), IFN-α (c), anti-ICOS (d), or CD28 (e). For each individual, the mean spot count of duplicate wells is
shown. Horizontal bars indicate the
means of all individual responses to the respective antigens. P values are given for means that are
significantly different from baseline values in (a). Symbols in (a) also apply to (b)–(e).
(a)(b)(c)(d)(e)Figure1(a) illustrates the low
baseline responses to NS3 protein and Pepset 4 pool (2.5±2.5 and 6.8±9.3
spots above medium, resp.) as frequently described in the literature in
the case of chronic infection [3–10]. The presence of IL-7/IL-15 during in
vitro T cell stimulation led to a significant ten-fold upregulation of
IFN-γ recall responses to NS3 protein (Figure 1(b); P  =  .02). Three of the patients also showed increased
responses to Pepset 4. However, due to
the wide range of spot numbers, the mean increase was not statistically
significant. Neither IFN-α  (Figure 1(c))
nor anti-ICOS (Figure 1(d)) had a general effect on HCV-specific
responses. Only HCV patients 1 and 7
showed an increased NS3 response upon IFN-α stimulation (P  =  .002 and P  =  .026, resp.). Costimulation with anti-CD28 (Figure 1(e)) showed
similar results compared to IL-7/IL-15 with a significant four-fold increase of
spot numbers (P  =  .021) after recall with NS3 protein. This is consistent with observations made by
Yonkers et al. and Ott et al. showing that HCV-specific T cells remain
responsive to CD28 stimulation during chronic infection [11, 13]. This consistency of results despite
significantly different patient age distributions in the aforementioned studies
also shows that patient age does not seem to influence the effects of in vitro costimulation.


## 3.3. In Vitro Costimulation Has No Effect on Antigen-Specific T Cell Responses in
HIV-Infected Patients

In order to
explore whether the aforementioned types of costimulation have a similar effect
in the case of HIV infection, we tested recall responses to three different HIV
peptide pools in 10 HIV-infected patients. Results are shown in Figure2.In vitro costimulation has no effect on
antigen-specific T cell responses in HIV-infected patients. PBMCs of 10 HIV-infected patients were tested in IFN-γ ELISPOT assays for their recall responses to HIV peptide pools 1, 2,
and 3 (as described in Section 2)
in the absence (a) or presence of IL-7/IL-15 (b), IFN-α (c), anti-ICOS (d), or
CD28 (e). For each individual, the mean
spot count of duplicate wells is shown. Horizontal bars indicate the means of all individual responses to the
respective antigens. Symbols in (a) also
apply to (b)–(e).
(a)(b)(c)(d)(e)Unlike in HCV, some of the patients
showed positive baseline responses to HIV peptides (Figure2(a)). However, none of the costimulatory reagents
could significantly enhance the overall response pattern, as shown in Figures 2(b)–2(e).  None of the mean responses to any of the
antigens was significantly elevated, regardless of the
(interindividually highly variable) baseline reactivity. Results were also identical in both
antiretrovirally treated and untreated patients, showing that antiretroviral
therapy did not seem to influence the effects of in vitro costimulation. This
decreased responsiveness of HIV-infected lymphocytes to costimulation has
previously been described by Borthwick et al. [20] and Trimble et al. [21], and
is attributed to the downregulation of costimulatory molecules such as CD28 on
T cells in the course of chronic HIV infection [22–24].  The precise mechanisms of this defect,
however, are still not understood. Our
data indicate that this effect may not be limited to CD28.


## 3.4. In Vitro Costimulation Causes False-Positive T Cell Responses to HCV and HIV
Antigens in Healthy Individuals

To date, there are
no conclusive data available regarding the effect of in vitro costimulation on HCV- and HIV-specific recall responses
in healthy individuals. This information
is crucial, though, in order to be able to properly interpret positive test
results obtained from assays using in
vitro costimulation. Therefore,
we specifically tested the same HCV and HIV antigens as above in 6 healthy
individuals (Figure3). If in vitro costimulation enhances
existing responses without altering disease specificity, it should not induce
positive responses in healthy individuals.In
vitro costimulation causes false-positive T cell responses to HCV and HIV
antigens in healthy individuals. PBMCs of 6 healthy individuals were
tested in IFN-γ ELISPOT assays for their recall responses to NS3 protein,
Pepset 4 peptide pool, and HIV peptide pools 1, 2, and 3 in the absence (a) or
presence of IL-7/IL-15 (b), IFN-α (c), anti-ICOS (d), or CD28 (e). For each individual, the mean spot count of
duplicate wells is shown. Horizontal
bars indicate the means of all individual responses to the respective
antigens. P values are given for
means that are significantly different from baseline values in (a). Symbols in (a) also apply to (b)–(e).
(a)(b)(c)(d)(e)As shown in Figure3, all four
methods of in vitro
costimulation (Figures 3(b)–3(e)) caused
marked responses to HCV and/or HIV antigens in several healthy
individuals. IL-7/IL-15 (Figure 3(b))
led to a strong positive response to Pepset 4 peptide pool (twenty-fold
increase above baseline; P  =  .026) as well as to HIV pool 1 (ten-fold
increase above baseline; P  =  .030). The mean frequency of NS3 reactive T cells was increased six-fold. Although this shift in mean frequency is not
statistically significant due to the broad range of anti-NS3 responses, the
changes for individual subjects are highly significant (P  =  .002 for
Healthy Control 1; P  =  .005 for Healthy Control 2).Similarly,
IFN-α also induced false-positive responses to NS3 (Figure 3(c)), as seen in
two individuals (P  =  .028 for Healthy Control 1; P  =  .021 for
Healthy Control 2). Interestingly, these
individuals had also displayed susceptibility to IL-7/IL-15 stimulation (Figure
3(b)). In vitro costimulation
with anti-ICOS agonist (Figure 3(d)) upregulated anti-NS3 responses to a lesser
degree. Healthy Control 4 responded with ~300 spots above baseline (P  <  .001). Remarkably, this individual had previously proven nonreactive to
both IL-7/IL-15 and IFN-α. The mean
frequency of Pepset 4 reactive T cells increased four-fold (P  =  .011)
upon ICOS stimulation.Finally,
as shown in Figure3(e), CD28 costimulation also caused false-positive test
results. Healthy Control 4 responded to
NS3 approximately three-fold above baseline (P  =  .006). Moreover, mean
spot counts for Pepset 4 were also increased significantly (P  =  .033).


## 4. Discussion

Efforts for in vitro T cell analysis attempt to
establish frequencies of antigen-reactive cells and their effector functions,
defined by the release of cytokines secreted upon antigen encounter. Tetramer staining for intracellular
cytokines, cytolytic molecules, and antigen-specific T cell receptors (TCRs) is
a technique frequently used for this purpose. It further allows the characterization of cell surface marker expression
by antigen-specific cells, distinguishing, for example, between central and
effector memory cells or between CD4 and CD8 cells [25].In
many cases, however, there is no true dominance of a particular peptide antigen. This has been described in HIV and recently
also in response to vaccinia virus [26]. For this reason, immune diagnostics in infectious diseases is shifting
towards the use of peptide pools as antigens. Since this approach is unfeasible in flow cytometry,  functional test systems are commonly used for
this purpose in which antigen directly activates the T cells and the resulting
cytokine secretion is detected. Cytokines can be measured in the cell supernatant by ELISA or directly
around the secreting cell using ELISPOT assays. ELISPOT assays typically are more sensitive compared to ELISA because
they are able to detect the cytokine production of each individual cell before
the cytokine is diluted into the supernatant, degraded by proteases, or
neutralized by receptors of bystander cells. As far as cell frequencies are
concerned, ELISPOT essentially provides the same information as intracellular
cytokine staining (ICS). However, the
detection limit of ELISPOT can be as low as 1/100 000 cells, ten times lower
than ICS [27]. Moreover, ELISPOT
measures the biologically relevant secretion of a cytokine as opposed to its
intracellular storage detected by ICS.  The intracellular presence of granzyme B or perforin,
for example, merely indicates that the cell has encountered antigen within the
past month [28]. In contrast, the actual
release of granzyme B or perforin indicates immediate antigen recognition by a
specific cell [1, 2].All
of these functional assays, however, have one major weakness in common. They provide accurate information on T cell
frequencies and function only if all specific T cells present in the test cell
population undergo full activation. For
example, recognition of an antigen presented by a dendritic cell (DC) along
with strong costimulation will result in full T cell activation. This lies  in
contrast with T cell activation by B cells which are known to be cells with
rather weak costimulatory capabilities [29]. In the latter case, only a fraction of antigen-specific T cells will be
fully activated. As a consequence, the
assay will mainly detect those cells that happen to make contact with a DC
instead of a B cell when sedimenting out of single-cell suspension onto the
bottom of the test plate. Since DCs are
less abundant in peripheral blood than B cells, the frequencies of
antigen-specific T cells will be considerably underestimated. A T cell would acquire different effector
functions depending on the type of APC presenting the antigen, and functional
assays would be strongly influenced by the prevalent types of APC populations
in the blood. Equally, conditions that
lead to APC depletion or inactivation, such as HIV and cancer, would leave many
antigen-specific T cells undetected, due to the lack of proper
stimulation. This is one of the reasons
why in chronic diseases such as HIV and HCV it has been notoriously difficult
to detect antigen-specific T cells in significant numbers.  Are T cells functionally impaired under such
conditions or is their activation hampered by limited numbers of APC? This has not been sufficiently studied
yet. For this reason, there have been
increasing efforts in the field to include costimulatory signals in T cell
activating cultures in order to overcome the deficiencies of the APC
compartment or of the T cells themselves.A
prototypic costimulatory molecule is CD28 [30], the only B7 receptor
constitutively expressed on naive T cells. In addition to ligation of TCR and
MHC antigen complex, binding of CD28 to either B7.1 or B7.2 on the APC leads to
upregulation of autocrine IL-2 secretion by the T cell. CD28 is commonly used for signal enhancement
in flow cytometry. In a previous study,
we found that it can also have “signal-enhancing” activity on recall
responses to tetanus, mumps, CMV, and EBV antigens in healthy individuals.In
the experiments reported here, we extended our previous studies and tested the
effects of different types of in vitro
costimulation on HCV- and HIV-specific recall responses not only in HCV- and
HIV-infected patients, but also in healthy individuals. Strikingly, we found that even in healthy
donors anti-CD28 induced significant responses to Pepset 4 antigens in addition
to a strong response to NS3 in one donor. While in HCV patients the results were weakly increased for NS3, in HIV
patients CD28 costimulation did not result in signal enhancement to any of the
antigens tested. Therefore, we conclude
that the use of anti-CD28 agonist provides no consistent benefit in either HIV
or HCV infection. In vitro costimulation with a combination
of anti-CD28 and anti-CD49d antibodies led to similar results (data not shown).We
also tested the use of IFN-α as a costimulatory reagent. IFN-α constitutes a “danger signal”
released by cells of the innate immune system when infected with virus or upon
TLR stimulation [31]. In our study, IFN-α caused false-positive responses to
NS3 in two of the healthy donors, while having little to no effect on responses
to any of the other antigens. In HIV
patients, the antigen-specific recall responses were even reduced in the
presence of IFN-α while in HCV patients they were increased to NS3 protein in
two of the patients. These data warrant
further studies and clearly show that the use of IFN-α for in vitro costimulation can result in
false-positive responses in healthy individuals while showing no useful effect
in either HCV or HIV infection.We
also tested agonistic anti-ICOS antibody as a costimulatory reagent.  Belonging to the CD28 family [30], anti-ICOS
showed similar effects to anti-CD28.  It
did not have significant effects on recall responses in HIV and HCV patients,
but caused false-positive responses to NS3 in healthy controls.The
combination of IL-7 and IL-15 has been used in in vitro assays to increase
assay sensitivity [12].  Particularly for
CD8 memory cells, both cytokines represent growth and differentiation
factors. Strikingly, the combination of
IL-7 and IL-15 induced strong responses in healthy controls to NS3 protein,
Pepset 4, and HIV pool 1. Increased
“antigen-specific” recall responses to both NS3 and Pepset 4 were
also seen in HCV patients. However, in
the light of the nonspecific stimulatory effect of IL-7/IL-15 in healthy
controls, these responses cannot be interpreted as specific. Finally, similar to IFN-α, the combination of IL-7/IL-15 led to a
marked reduction of cytokine responses to all three HIV peptide pools in HIV
patients. It is unclear why HIV-infected
individuals respond so differently compared to HCV patients and healthy
controls. Possible reasons include the
reduced T cell function and susceptibility to costimulation as a consequence of
HIV infection as previously described for CD28 [22–24].


## 5. Conclusion

Overall, our
data show that in vitro costimulation
with IL-7/IL-15, IFN-α, anti-ICOS, and anti-CD28 is no “magic bullet”
providing simple solutions for improved T cell diagnostics. There are two criteria that a method of in vitro costimulation has to fulfill
in order to qualify as clinically and/or scientifically useful, reliably
enhancing antigen responses in a disease-specific manner without causing
false-positive responses in healthy individuals. Our results show that essentially none of the
stimulatory molecules tested met these criteria. There are two possible interpretations of
these results. On the one hand,
insufficient costimulation may not be the limiting factor in HCV or HIV
patients, which explains why additional costimulation does not help to reveal
more specific activity. The other
possibility is that the functional defects present in diseases like HIV and HCV
are too severe—either on the T
cell or the antigen presentation side—to be overcome by
in vitro costimulation.



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*Source: 590941-2008-07-21.xml*