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Prepublished online as a Blood First Edition Paper on November 14, 2002; DOI 10.1182/blood-2002-07-2103.
IMMUNOBIOLOGY
From the Vaccine Research Center and the Laboratory of
Immunoregulation, National Institute of Allergy and Infectious Diseases
(NIAID), National Institutes of Health (NIH), and the Experimental
Transplantation and Immunology Branch, NCI, NIH, Bethesda, MD; and the
Departments of Pathology and Neurology, University of Texas,
Southwestern Medical Center, Dallas.
Virus-specific CD8+ T-cell responses play a pivotal
role in limiting viral replication. Alterations in these responses,
such as decreased cytolytic function, inappropriate maturation, and limited proliferative ability could reduce their ability to control viral replication. Here, we report on the capacity of HIV-specific CD8+ T cells to secrete cytokines and proliferate in
response to HIV antigen stimulation. We find that a large proportion of
HIV-specific CD8+ T cells that produce cytokines in
response to cognate antigen are unable to divide and die during a
48-hour in vitro culture. This lack of proliferative ability of
HIV-specific CD8+ T cells is defined by surface expression
of CD57 but not by absence of CD28 or CCR7. This inability to
proliferate in response to antigen cannot be overcome by exogenous
interleukin-2 (IL-2) or IL-15. Furthermore, CD57 expression on
CD8+ T cells, CD4+ T cells, and NK cells is a
general marker of proliferative inability, a history of more cell
divisions, and short telomeres. We suggest, therefore, that the
increase in CD57+ HIV-specific CD8+ T cells
results from chronic antigen stimulation that is a hallmark of HIV
infection. Thus, our studies define a phenotype associated with
replicative senescence in HIV-specific CD8+ T cells,
which may have broad implications to other conditions associated with
chronic antigenic stimulation.
(Blood. 2003;101:2711-2720) CD8+ T cells are crucial to the
recognition and clearance of virus-infected cells.1,2
Fully functional CD8+ T cells have the ability to
proliferate and mediate antiviral activity through cytokine and
chemokine secretion, Fas/Fas ligand interactions, and/or
perforin/granzyme-mediated cell lysis after recognition of cognate
antigen. When naive CD8+ T cells are activated during a
primary viral infection, they proliferate and become effector T cells
that fulfill these functions.2,3 Following clearance of
virus, the majority of the virus-specific CD8+ T cells die,
and few memory CD8+ T cells remain to combat subsequent
infections with further rounds of proliferation and elaboration of
effector functions.
Studies have suggested that chronic stimulation of T cells, such
as that which occurs with rheumatoid arthritis,4-6
multiple myeloma,7 cytomegalovirus (CMV) and HIV
infections,8,9 and following bone marrow
transplantation10 can result in the development of
CD8+ T cells that are capable of cytokine secretion yet
incapable of cell division. Although the provenance and exact phenotype of such CD8+ T cells remain unclear,10-16 such
a failure to proliferate is generally attributed to replicative
senescence resulting from continual stimulation by antigen and/or
cytokine. Indeed, it has been suggested that replicative
senescence17-21 or "clonal
exhaustion"22-24 of HIV-specific T cells may underlie
the inability of T-cell immunity to suppress virus adequately. Other
studies have suggested that deficiencies in HIV-specific
CD8+ T-cell function may arise from insufficient
CD4+ T-cell help25-28 or specific signaling
and cytotoxic functional defects.25,29,30
The phenotypes associated with replicatively senescent CD8+
T cells are not well defined31,32 but are generally
attributed to lack of CD28 or expression of CD577,30,33-36
and are thought to result in the inability of these CD8+ T
cells to proliferate.37,38 These T cells commonly are
found in individuals with chronic immune
activation,4,6-10,39,40 and they increase in frequency
with age.41,42 We examined the relationship between CD57
expression and 2 functional aspects of HIV-specific, CMV-specific, and
other CD8+ T cells: their ability to produce cytokines and
to proliferate in response to stimulation by cognate antigen. We found
that CD57+ HIV-specific CD8+ T cells,
irrespective of CD28 or CCR7 expression, produce IFN- Study subjects
Peptides
Identification of HIV-specific CD8+ T cells (6-hour assay) Peripheral blood mononuclear cells (PBMCs) were isolated and, in some instances, viably cryopreserved until later use. Stimulation was performed on fresh or frozen PBMCs as previously described.43,44 In every experiment a negative control (anti-CD28/CD49d) was included to control for spontaneous production of IFN- , as well as a positive control (Staphylococcus
enterotoxin B (SEB) 1 µg/mL final, Sigma, St Louis, MO) to ensure
that cells were responsive. Cultures were incubated for 1 hour at
37°C, followed by an additional 5 hours in the presence of
Brefeldin-A (BFA) (1 µg/mL, Sigma). CD8+ T cells that
produce effector cytokines following antigenic-specific stimulation are
contained within and are thought to represent the tetramer-binding
CD8+ T cells.43,45-47
Antigen-specific proliferation (48-hour assay) PBMCs were initially stained with carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) as previously described.48 These PBMCs were suspended in media supplemented with IL-2 (10 U/mL, Sigma) or IL-15 (100 ng/mL, R&D Systems, Minneapolis, MN) and were then stimulated with HIV peptide pools as described.43,44 Cells were then incubated for 36 hours at 37°C followed by an additional 12 hours in the presence of Brefeldin-A (10 µg/mL, Sigma). Cells were then stained for flow cytometric analysis. The percentage of HIV-specific CD8+ T cells was determined as in the previous paragraph. The proportion of CD8+ T cells that showed CFSE dilution (division) at 48 hours was divided by 2 (a cell division represents a 2-fold increase in frequency, hence the percentage of proliferated CD8+ T cells must be halved) and then divided by the 6 hours' response; for instance ([1/2 48 hour divided response]/6 hours response). This number represents the percentage of antigen-specific CD8+ T cells that had divided in response to antigenic stimulation.Mitogen and superantigen stimulations For superantigen stimulations SEB (Sigma) was used at 1 µg/mL. For mitogenic stimulations phytohemagglutinin (PHA) (Sigma) was used at 5 µg/mL. PBMCs stimulated with SEB were cultured for 4 days prior to immunofluorescent staining, and PHA-stimulated PBMCs were cultured for 2 days prior to propidium iodide staining. For apoptosis studies, SEB-stimulated PBMCs were cultured for 24 hours in the presence of activated caspase 3 binding peptide Z-VK(biotin)D(OMe)-FMK at 43 µg/mL (Enzyme Systems Products, Livermore, CA).Immunofluorescence staining Stimulated PBMCs were washed and then surface stained with directly conjugated antibodies to CD3 and other surface markers (Becton Dickinson Immunocytometry Systems (BDIS), San Jose, CA) for 20 minutes on ice. The cells were washed and fix/permeabilized (fixation/permeabilization solution [BDIS]), and stained with directly conjugated antibodies to IFN- or other intracellular molecules (as required for specific experiments), and resuspended in
1% paraformaldehyde in phosphate-buffered saline.
Flow cytometric analysis Using a FACScalibur flow cytometer (BDIS), 6-parameter flow cytometric analysis was performed. Fluorescein isothiocyanate (FITC) or CFSE, PE, PerCP, and allophycocyanin (APCs) were used as the fluorophores. At least 100 000 live CD3+ lymphocytes were collected. Twelve-parameter flow cytometric analysis was performed using a modified FACSDIVA flow cytometer (BDIS). Alexa 430, FITC, PE, Texas-Red PE, Cy5PE, Cy5.5PE, Cy7PE, APC, Cy7APC, and Alexa 594 were used as fluorophores. The list-mode data files were analyzed using PAINT-A-GATE software (BDIS) and FlowJo software (Tree Star, San Carlos, CA). CFSE distributions were analyzed using FlowJo software.Cell sorting Cell sorting was accomplished using a FACSDiva cell sorter (BDIS). FITC, PE, Cy5PE, and APC were used as the fluorophores. At least 10 000 cells were sorted for PCR analysis, and at least 106 cells were sorted for telomere length and cell-division analysis.NK cells were magnetically sorted by a positive selection method with use of anti-CD16 antibodies conjugated to microbeads (Miltenyi Biotec, Auburn, CA). T-cell receptor excision circle analysis Quantification of T-cell receptor excision circle (TREC) in sorted CD8+ T cells was performed by real-time quantitative PCR by means of the 5' nuclease (TaqMan) assay with an ABI7700 system (PerkinElmer, Norwalk, CT) as previously described.49,50Telomere length analysis Telomere length analysis was performed using peptide nucleic acids (Dako, Carpinteria, CA) as previously described.51,52 Analysis was accomplished by comparing mean fluorescence intensity of the telomere channel between different populations that were all gated for similar DNA content (propidium iodide fluorescence) between samples.Statistical analysis Correlations were performed by Spearman rank correlation, and statistical significances were performed by Wilcoxon matched pairs test using Prism 3.0 software (San Diego, CA).
Proliferative defect in HIV-specific CD8+ T cells As functional defects of antigen-specific T cells in humans recently have been described in HIV infection, we analyzed antigen-specific CD8+ T-cell responses in healthy and HIV-infected subjects by IFN- secretion and proliferation by flow
cytometry. Figure 1A illustrates this
using PBMCs from a healthy HLA-A2+ individual (subject 31),
which we labeled with CFSE and stimulated with the HLA-A2-restricted
CMV pp65 peptide (NLVPMVATV). After 6 hours, 27% of CD8+ T
cells produced IFN- but had not divided. By 48 hours, 13% of the
CD8+ T cells had divided and continued to produce IFN-.
The percentage of responding CD8+ T cells and their ability
to proliferate were assessed also by tetramer analysis to assure that
the IFN--producing CD8+ T cells adequately represented
the antigen-specific CD8+ T cells (data not shown). Despite
this relatively high percentage of antigen-specific CD8+ T
cells, this response clearly demonstrated that the described assay
could be used to monitor proliferation in response to
antigenic stimulation.
To examine cytokine secretion and proliferation in HIV-specific
CD8+ T cells, we stimulated PBMCs from 11 HIV-infected
subjects with a pool of overlapping 15-mer peptides encompassing HIV
gag.43,45 In contrast to the CD8+ T-cell
proliferation observed for CMV-specific CD8+ T cells from
subject 31, a high proportion of HIV-specific CD8+ T cells
did not proliferate (Table 2). Three
patterns of responsiveness were apparent, as summarized in Figure 1B-D.
HIV gag-specific CD8+ T cells from subject 15 were capable
of proliferation and continued to produce IFN-
HIV-specific CD8+ T cells that do not proliferate are defined by CD57 expression Having determined the proliferative potential of the HIV-specific CD8+ T cells (Figure 1; Table 2), we sought to define phenotypic markers that might differentiate proliferating from nonproliferating HIV-specific CD8+ T cells (Table 2).As shown in Table 2, CD57 was the only surface marker we found that
alone defined the proliferative ability of HIV-specific CD8+ T cells (Figure 2; Table
2). In addition to surface molecules listed in Table 2, we also studied
expression patterns of CD152, CD11b, CD45RA, CD95, and CD45RO by
HIV-specific CD8+ T cells but found that these molecules
did not differ between T cells that proliferated and those that did
not. A recent study suggested that CCR7
We further confirmed the association between CD57 expression and
proliferative inability in 2 ways. First, in subject 15, whose
CD8+ T cells proliferated in response to HIV gag, all the
IFN- The presence of nondividing CD57+CD8+ T cells
specific for HIV gag correlated with that of
CD57+CD8+ T cells specific for HIV pol, env,
and nef (R = 0.95, P < .0001), suggesting that the
generation of such nondividing cells is related to the response to the
whole pathogen rather than to individual antigens (Figure
3A). In addition, the fraction of
HIV-specific CD8+ T cells that are CD57+
remained constant (the absolute number of CD57+
HIV-specific CD8+ T cells decreased) following highly
active antiretroviral therapy (data not shown). Figure 3B
demonstrates that CD57 expression by CD8+ T cells specific
for antigens other than HIV is only marginally correlated with
expression of CD57 by HIV-specific CD8+ T cells
(R = 0.58, P = .003). Whether this disparity is a result of differential cytokine expression by a significant proportion of
HIV-specific CD8+ T cells or an underestimation of
responses due to sequence differences or HIV accessory gene-specific
CD8+ T cells remains unclear. However, it is clear that
HIV-specific CD8+ T cells can contribute substantially (up
to 80%) to the total CD57+CD8+ T-cell
population in HIV-infected individuals.
We also investigated the effect of CD57 expression on all T-cell subsets by stimulating CFSE-labeled PBMCs from 2 HIV-infected individuals and 5 HIV-uninfected subjects with superantigen. No proliferation was observed within the CD57+CD8+ or CD57+CD4+ T cells. Furthermore, we studied proliferation of natural killer (NK) cells following stimulation with IL-2 and similarly found no proliferation within the NK cells, which expressed CD57 (data not shown). Hence, lack of proliferation by cells expressing CD57 is not restricted to HIV-specific CD57+CD8+ T cells but is a property of all T cells and NK cells. It has been described that CD57 and CD28 expression by CD8+
T cells are mutually exclusive.7,33,36,53 However, by
multiparameter flow cytometric analysis of memory CD8+ T
cells from 6 healthy subjects, we found that 4 populations of
CD8+ T cells were evident by CD28 and CD57 expression
(Figure 4). In all subjects there were
considerable levels of CD8+ T cells that were either
CD57+CD28+ or
CD57
It has been suggested that memory CD8+ T cells that
"revert" to a CD45RA+ phenotype represent a population
of terminally differentiated fully competent "effector memory" T
cells.30,54,55 In order to define further the
CD57+CD8+ T-cell population, we sought to
examine the relationship between expression of the markers that have
been used to define naive, "central memory," and effector memory
populations: CD57, CD27, CD28, CCR7, CD62L, CD45RO, and CD45RA. Figure
5 shows how we performed this using
10-color flow cytometry. Figure 5A shows that most of the effector
memory (CD45RA+CD27
CD57+ CD8+ T cells are characterized by replicative senescence The lack of proliferation of CD57+ HIV-specific and other CD8+ T cells suggests that they may have reached replicative senescence. Indeed, it has been shown that CD28 CD8+ T cells, the majority of which we
have shown are a subset of CD57+CD8+ T cells,
have shortened telomeres.19,41,56 To address this possibility, we examined the telomere lengths of CD57+ and
CD57 memory and naive CD8+ T cells in 3 healthy volunteers (representative plot in Figure 6A). The
CD57+CD8+ T cells had shorter telomeres than
either CD57 memory or naive CD8+ T cells.
Taken together, these results indicate that the
CD57+CD8+ T-cell population, as a whole, had
undergone more cell divisions than the
CD57CD8+ T-cell population. However, the
observed differences in telomere length between each subset, although
consistent from subject to subject, were small and did not reach
statistical significance.
In order to quantify proliferation differences between
CD57+ and memory CD57 Previous data have suggested that replicatively senescent T cells can
be defined by loss of CD28 expression.19,56,58 Because we
found discordance in proliferative ability within populations defined
by CD57 and CD28, we sought to determine the proliferative history of
each population defined in Figure 4. Hence, we sorted by flow
cytometry CD8+ T cells that were naive
CD45RO CD57+ CD8+ T cells are sensitive to induction of apoptosis The observation that CD57+ HIV-specific CD8+ T cells fail to proliferate and can no longer be detected by IFN- after 48 hours in vitro stimulation suggests that
these T cells might be undergoing activation-induced cell death.
Alternatively, they may remain in culture but fail to produce IFN-
at 48 hours. To address these possibilities, we used an HLA-A2 tetramer
complexed with an HLA-A2-restricted HIV gag peptide (amino acid
sequence SLYNTVATL) in an HLA-A2+ subject (subject 8) with
a response to this epitope and found that 51% of tetramer-binding
CD8+ T cells were CD57+. After stimulation with
SLYNTVATL peptide followed by a 48-hour incubation, only 11% of the
tetramer-binding CD8+ T cells were CD57+ (data
not shown), suggesting that the CD57+ tetramer binding
CD8+ T cells were no longer present in the culture.
To determine if CD57+CD8+ T cells were more
prone to undergo activation-induced cell death, we sorted
CD57+ and CD57
The cytokine IL-15 has been shown to reduce apoptosis59,60
and promote proliferation of HIV-specific CD8+ T
cells.61 However, we found that although IL-15
dramatically increased the percentage of proliferating
CD57
We have shown that HIV-specific CD8+ T cells that
express the surface molecule CD57 are incapable of proliferating after
antigen-specific stimulation in vitro and undergo activation-induced
apoptosis. Furthermore, this behavior is not associated with loss of
CD28 expression. Some studies have suggested that CD57 and CD28
expression are mutually exclusive in memory T-cell
populations.7,35,53,56 However, we found that
CD57+CD28+ and
CD57 CD57 expression is not limited to HIV-specific CD8+ T cells but is associated with the same properties among all T cells and NK cells. These data indicate that cells of both the adaptive arm as well as the innate arm of the immune system are capable of reaching a state of replicative senescence. From the very low TREC levels and the shortened telomere lengths in
CD57+CD8+ T cells, it is clear that this
population has undergone more rounds of replication in vivo than
CD57 While previous studies have suggested an inability of HIV-specific
CD8+ T cells to mature to
CD45RA+CCR7 Furthermore, CD8+ T cells belonging to the effector memory
population are defined as
CD45RA+CCR7 HIV-specific CD8+ T cells can contribute a large proportion (up to 80%) of the total CD57+ T cells, and there is a statistically significant correlation between the percentage of CD57+ HIV gag-specific CD8+ T cells and CD57+CD8+ T cells specific for other HIV antigens. Others have reported increased CD57 expression in persons with chronic CMV infection,9,36,64 even suggesting that all CD57+CD8+ T cells are specific for CMV antigens.65 Thus, the generation of replicative senescence of CD8+ T cells may occur as a result of repetitive antigenic stimulation in vivo by chronic persistent viruses such as CMV and HIV. Studies aimed at correlating HIV-specific CD8+ T-cell
responses with markers of HIV disease have resulted in different
conclusions.27,45,66-68 Whereas there is a negative
correlation between viral load and specific populations of bulk
CD57+CD8+ and
CD28 While the mechanism by which CD57 expression exerts these described phenotypes upon lymphocytes is unknown, and if CD57 is even, itself, involved in apoptosis and lack of proliferation is unknown, the proliferative defects and apoptotic nature observed within HIV-specific CD8+ T cells can be predicted by expression of CD57 on cells that have previously undergone multiple rounds of cell division. The presence of these cells, however, does not reflect a defect particular to the immune response to HIV or an effect limited to any particular virus, but simply reflects the normal consequence of persistent immune activation. Taken together, our data shed light on the functionality and provenance of CD57+ lymphocytes. Hence, in HIV disease, the presence of proliferation-incompetent HIV-specific CD8+ T cells is the result, not the cause, of uncontrolled viral replication. Although several conditions characterized by chronic antigenic stimulation may result in such an immune state, this underlying pathogenesis is a hallmark of untreated HIV infection.
Submitted July 15, 2002; accepted November 8, 2002.
Prepublished online as Blood First Edition Paper, November 14, 2002; DOI 10.1182/blood-2002-07-2103.
Supported by NIH grant AI49990 (N.J.K.) and RO1 AI47603 (N.J.K.), and the Distinguished Young Researcher Award from the UT Southwestern President's Research Council (N.J.K.).
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 U.S.C. section 1734.
Reprints: Richard A. Koup, Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, 40 Convent Dr, Bethesda, MD 20892; e-mail: rkoup{at}mail.nih.gov | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Abstract
Introduction
) and
CD57
)
(B). HIV-specific CD57+ (
CD28+CD57
CD28