Institute of Medical Microbiology and Immunology, University of Copenhagen, The Panum Institute, 3C Blegdamsvej, DK-2200 Copenhagen N, Denmark1
Author for correspondence: Allan Randrup Thomsen. Fax +45 35 32 78 91. e-mail A.R.Thomsen{at}immi.ku.dk
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Abstract |
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Introduction |
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Methods |
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Virus.
All strains of lymphocytic choriomeningitis virus (LCMV) used in this study were produced, stored and quantified as previously described (Nansen et al., 1999 ). Infected mice received the virus as an intravenous (i.v.) injection of 0·3 ml; inoculation by this route results in a non-lethal infection (Nansen et al., 1999
). Vesicular stomatitis virus (VSV) of the Indiana strain (originally provided by K. Berg of this institute) was given as an i.v. dose of 106 p.f.u. This dose is non-lethal in immunocompetent mice, but induces a potent CD8+ T cell response (Andreasen et al., 2000
).
Cell preparations.
Single-cell suspensions of spleen cells were obtained by pressing the spleen through a fine steel mesh. Peritoneal cells were obtained by lavage with 5 ml of cold Hanks' balanced salts solution.
Monoclonal antibodies for flow cytometry.
The following mAbs were purchased from PharMingen as rat anti-mouse Abs: CyChrome (Cy)-conjugated anti-CD4 and anti-CD8a, FITC-conjugated anti-CD49d [common -chain of lymphocyte Peyers patch adhesion molecule-1 and very late Ag-4 (VLA-4)], FITC- and PE-conjugated anti-IFN-
, PE-conjugated anti-IL-2 and matched isotype controls.
MHC/peptide tetramers for flow cytometry.
H-2Ld/np118126, H-2Db/gp3341 and H-2Db/np396404 tetramers were obtained through the National Institute of Allergy and Infectious Disease tetramer facility and the National Institutes of Health AIDS Research and Reference Reagent Program.
Flow cytometric analysis.
Staining for flow cytometry was performed as described previously (Andreasen et al., 2000 ). Briefly, 1x106 cells were stained with directly labelled mAbs in FACS medium (PBS containing 10% rat serum, 1 % BSA and 0·1% NaN3) for 20 min in the dark at 4 °C. After washing twice, cells were fixed with 1% paraformaldehyde. For tetramer staining (Christensen et al., 2001
), cells were incubated with the tetramers for 30 min at 4 °C, at which time Abs for surface labelling were added, and the cells were then incubated for a further 30 min. To detect intracellular IFN-
and IL-2, splenocytes were cultured at 37 °C in a 96-well round-bottomed microtitre plate at a concentration of 2x106 cells per well in a volume of 0·2 ml complete RPMI medium supplemented with murine recombinant IL-2 (50 U/ml), monensin (3 µM) and peptide (Andreasen et al., 2000
; Christensen et al., 2001
). The peptides were used at a concentration of 0·1 µg/ml (LCMV gp3341, np396404 and np118126) or 1 µg/ml (LCMV gp6180 and VSV np5259). After 56 h of culture, the cells were washed once in FACS medium (PBS containing 1% BSA, 0·1% NaN3 and 3 µM monensin) and subsequently incubated with relevant surface antibodies in the dark for 20 min at 4 °C. Cells were washed twice in PBS with 3 µM monensin, resuspended in 100 µl of PBS, and 100 µl 2% paraformaldehyde in PBS was added. After 30 min of incubation in the dark at 4 °C, cells were washed in FACS medium without rat serum but containing 3 µM monensin and subsequently resuspended in PBS with 0·5% saponin. After 10 min of incubation in the dark at 20 °C, cells were pelleted and resuspended in PBS with 0·5% saponin and relevant antibodies. After incubation for 20 min at 4 °C, cells were washed twice in saponin. Samples were acquired on a FACSCalibur (Becton Dickinson), and at least 105 mononuclear cells were gated using a combination of forward angle and side scatter to exclude dead cells and debris. In some experiments, T cells were enriched by negative selection of B cells and FcR+ cells using Dynabeads (Dynal) prior to analysis. Data were analysed using Cell-Quest software (Becton Dickinson).
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Results |
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Chronic LCMV infection prevents normal CD8+ T cell differentiation
The above results indicate that IL-2-producing CD8+ T cells are generated as part of the differentiation that takes place during normal development of a CD8+ T cell response. However, what would happen if antigen persisted at a high level? To study this, B6 mice were infected with a high dose of the rapidly replicating clone 13 LCMV isolate. Following high dose infection with this LCMV variant, wild-type mice develop a chronic infection leading to exhaustion of the CD8+ T cell response (Zajac et al., 1998 ). The CD8+ T cell response was evaluated 6, 8, 10 and 20 days after infection, and the capacity of the CD8+ T cells to produce IL-2 and IFN-
was compared with that in mice infected with an immunizing dose of the more slowly replicating LCMV Armstrong strain. These virus strains express the same T cell epitopes, but LCMV Armstrong infection induces a transient, acute infection and no exhaustion is observed (Nansen et al., 1999
). The responses to both gp3341 and np396404 were studied; however, since essentially similar patterns were obtained, only data for gp3341 is presented.
As reported earlier (Zajac et al., 1998 ), clonal expansion was markedly reduced in clone 13-infected mice compared with Armstrong infected mice as evidenced by lower spleen cell numbers and fewer CD8+ T cells. More importantly in the present context, marked qualitative differences were already observable in the early phase of infection (Table 1
). Thus, on day 8 p.i., a high frequency of IFN-
-producing CD8+ T cells were found in Armstrong-infected mice, and about 10% of these cells also produced IL-2. In contrast, few if any IL-2-producing cells were detected in clone 13-infected mice. These mice did, however, contain nearly the same fraction of virus-specific (tetramer+) CD8+ T cells, some of which produced IFN-
(albeit at a reduced level, see Fig. 5
). Here the difference in the ratio of IFN-
+ to tetramer+ cells in the two groups should be noted: because the gp3341 peptide contains both a Db- and a Kb-restricted immunodominant epitope (Hudrisier et al., 1997
), more IFN-
+ than Db tetramer+ cells are always found following immunizing infection; in contrast, due to partial anergy the ratio between these phenotypes is 1:1 or less in clone 13-infected mice. Ten days after infection, the relative frequency of IL-2-producing CD8+ T cells was increased in Armstrong-infected mice, while the frequency of these cells remained low in clone 13-infected mice (representative plots are presented in Fig. 5
), with no cells producing both cytokines. At this time point, Armstrong-infected mice had cleared their infection while clone 13-infected mice contained high levels of virus (spleen: 3·5x107 p.f.u./g, lungs: 5x107 p.f.u./g; mean of four mice). Twenty days after infection, the ability to produce IFN-
was also almost lost in clone 13-infected mice, which still harboured high levels of virus. Thus, in contrast to the situation following acute immunizing infection, no IL-2-producing CD8+ T cell subset is established in chronically infected mice and at no time did we find a subset producing both IFN-
and IL-2. To evaluate fully the functional implications of this finding, the number of CD4+ cells producing IL-2 was assessed 6, 8 and 10 days after infection; at no time point did we detect a significant number of IL-2-producing CD4+ T cells in clone 13-infected mice, while numbers peaked at about 1·5x106 cells 8 days after infection with Armstrong virus.
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Discussion |
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In the present report, we have studied the ability of CD8+ T cells to produce IL-2 using flow cytometry to quantitate and characterize cytokine-producing cells. We found that following transient, immunizing infection, up to 1020% of antigen-specific CD8+ T cells eventually acquired the ability to produce IL-2. This frequency was lower than for a corresponding population of CD4+ T cells, but because of the high number of activated CD8+ T cells generated in the context of an antiviral response, this makes CD8+ T cells the prominent IL-2-producers during viral infection. This holds true even though there was a trend for a subset of CD4+ T cells to produce higher levels of IL-2 (as judged from their fluorescence signal) than any CD8+ T cells (see, for example, Fig. 1). We also found that CD4+ T cell-deficient MHC class II -/- mice initially responded with an unimpaired CD8+ T cell response. Thus, IL-2 production by CD8+ T cells may explain the difference in the level of immunodeficiency in IL-2- and CD4+ T cell-deficient mice, as well as the capacity of the latter mice to support an almost normal initial clonal expansion and differentiation within the CD8+ T cell subset. Due to the present lack of a specific surface marker to distinguish IL-2-producing cells, it is not possible to test this assumption directly by specific depletion of this subset. However, a functional importance of these cells is supported by previous observations indicating that time to rejection of MHC class I disparate skin grafts correlates directly with precursor frequency of IL-2-producing CD8+ T cells as assessed by limiting dilution (Rosenberg et al., 1986
).
Interestingly, our study also describes various functional phenotypes normally found within a population of in vivo primed CD8+ T cells with the same epitope specificity. Furthermore, the composition of the population with regard to these phenotypes provides critical information regarding the functional status of that CD8+ T cell population. In this respect, our results confirm and extend early studies (Gallimore et al., 1998 ; Ehl et al., 1998
) indicating that CD8+ T cell differentiation is critically affected by the virus load early after infection. Furthermore, the results obtained in class II -/- mice indicate that CD4+ T cells directly or indirectly are required to sustain the composition normally established after immunizing infection. Therefore, based on these results, we propose the following model for CD8+ T cell differentiation. During acute viral infection, most primed CD8+ T cells have the capacity to produce IFN-
, as previously noted by others (Butz & Bevan, 1998
; Murali-Krishna et al., 1998
). A small subset of these cells (typically
5% for immundominant epitopes) also has the capacity to produce IL-2. With clearance of the infection and transition into the memory phase, the relative frequency of cells producing both cytokines increases (a result of antigen clearance?). The latter finding fits well with existing data (Slifka & Whitton, 2000
) on coproduction of TNF-
and IFN-
, which also increase with time, and may suggest that under normal conditions there is development towards preferential maintenance of a multipurpose cell type (Veiga-Fernandes et al., 2000
). A similar pattern seems to exist for CD4+ T cells. In line with the assumption that coproducing cells represent a more advanced stage of differentiation, we also saw enrichment of this phenotype among effector cells that had migrated to a non-lymphoid organ site (Masopust et al., 2001
). Probably, the presence of IL-2-producing cells in the periphery would increase the ability of memory CD8+ T cells to function autonomously in case of rechallenge.
In contrast to transient, immunizing infection, differentiation of IL-2-producing CD8+ T cells appears to be inhibited under conditions that lead to a high initial virus load. In this case, the capacity to synthesize IFN- is also rapidly impaired, and most antigen-specific cells are eventually deleted (Zajac et al., 1998
). Since, in addition, no IL-2-producing CD4+ T cells could be detected in this case, lack of IL-2-producing CD8+ T cells may contribute to the total collapse of the primary antiviral T cell response.
If the initial virus load is not increased but antigen persists in the absence of CD4+ T cells (as in MHC class II -/- mice), IL-2-producing CD8+ T cells are initially generated, but cannot be sustained. A requirement for CD4+ T cells in regulating the functional state of virus-specific CD8+ T cells has been reported before (Zajac et al., 1998 ). However, in our case the exhaustive process is more gradual and less complete, allowing a better separation of stages in this process. Thus, in class II-deficient mice infected with LCMV Traub, primary CD8+ T cell expansion and differentiation appears normal. Gradually and starting before the rebound of viraemia in these mice (Thomsen et al., 1996
; unpublished observations), primed CD8+ T cells become impaired in their capacity to synthesize cytokines, first IL-2 and later also IFN-
. Since IFN-
is pivotal for virus control (Bartholdy et al., 2000
), reduced capacity to produce this cytokine may contribute to the high virus load found in class II -/- mice at late time points (Thomsen et al., 1996
). However, of particular interest in the present context is the finding that IL-2 production is lost very early in the process. Whether this reduction in IL-2 production by CD8+ T cells during the memory phase is of functional importance is not clear (Ke et al., 1998
; Ku et al., 2000
). Some evidence suggests that IL-2 may, in fact, negatively regulate memory CD8+ T cell numbers (Ku et al., 2000
). However, this may hold true only for homeostatic proliferation (no antigen present), a situation clearly distinct from that associated with a chronic viral infection. Indeed, the fact that we found no compensatory increase in the number of virus-specific CD8+ T cells present in class II -/- mice, despite a clearly increased virus load at day 90 p.i., could be taken to suggest some impairment of antigen-driven proliferation, even at this stage. More importantly, no secondary CTL response is generated when these cells are restimulated in irradiated recipients (Thomsen et al., 1996
), demonstrating the severely impaired function of these cells. Therefore, even if IL-2 has no direct functional role during chronic infection, our findings indicate that the failure to detect IL-2-producing CD8+ T cells constitutes an early and sensitive marker for a dysfunctional CD8+ T cell subset. Thus, it would seem that analysing IL-2 production by a population of virus-specific CD8+ T cells may provide important insight into the functional state of that population, particularly at times when exhaustion is incomplete or partial. In this context, it is relevant to note that there are published data (Itoh & Germain, 1997
) indicating a hierarchical pattern of signalling thresholds for cytokine responses, and that production of IL-2 requires a stronger signal than does IFN-
production. This could explain why IL-2 production is a more sensitive parameter when evaluating the functional integrity of a population of antigen-specific CD8+ T cells. With regard to the clinical significance of these findings, it should be mentioned that impaired signalling has been demonstrated for human immunodeficiency virus (HIV)-specific CD8+ T cells and reduced IFN-
production is found in late-stage patients (Lieberman et al., 2001
).
An important issue not resolved by this study is the causeeffect relationship between impaired virus control and CD8+ T cell dysfunction. Indeed, this is likely not to be a simple unidirectional relationship but rather a reciprocal interaction. While a reduced capacity to produce IFN- undoubtedly contributes to an increased virus load (Bartholdy et al., 2000
), it is also evident that strong and/or sustained antigenic stimulation leads to T cell dysfunction (Moskophidis et al., 1994
; Gallimore et al., 1998
; Ehl et al., 1998
). Thus, extended antigen stimulation could trigger a cascade of events, which at high levels of antigen result in rapid deletion of most antigen-specific cells, as seen in clone 13-infected mice. When antigen persists at lower levels, as observed in class II -/- mice, the process is slower and a subset of the cells (those of the highest affinity?) and/or certain functions are primarily affected. In this situation, CD4+ T cells may be important either to mediate the conditioning of APCs needed to sustain CD8+ effector function (Andreasen et al., 2000
; den Boer et al., 2001
; Sarawar et al., 2001
) or because CD4+ T cells are required to reduce virus levels to a point that will not over time exhaust the CD8+ T cells (Moskophidis et al., 1994
; den Boer et al., 2001
). These possibilities are not mutually exclusive; however, the present finding that CD8+ T cell dysfunction starts developing while the virus load is still low (Thomsen et al., 1996
) suggests that impairment of virus control is not the sole mechanism involved. Supporting this view, an accelerated decline of CD8+ T cell memory is also observed in CD4-deficient mice infected with vaccinia virus (von Herrath et al., 1996
), which is a cytolytic virus very unlikely to persist at significant levels in the host. Whatever the underlying mechanism, the present results indicate that the limiting function of CD4+ T cells is not merely production of IL-2, as CD8+ T cells seem fully capable of producing their own IL-2 given the right circumstances.
In conclusion, our results reveal that CD8+ T cells are prominent producers of IL-2 throughout a viral infection. The generation of this subset is delayed relative to the majority of IFN- producers, but may still be sufficiently rapid to be of functional relevance during the primary response (compare the difference in severity of the immune deficiency in CD4-deficient mice and IL-2 knockouts). As primed CD8+ T cells move into the memory phase, an increasing fraction also synthesize IL-2, and failure to complete this development (lack of CD4-dependent signals, high virus load) signifies an unstable situation associated with impaired capacity to control the infection. Why memory differentiation is linked to the appearance of cells with the capacity to produce IL-2 is not clear at the moment, but a similar association has recently been observed for CD4+ T cells (Saparov et al., 1999
), suggesting that this may be a general phenomenon. Therefore, analysing the ability of an antigen-specific T cell population to produce cytokine, and in particular IL-2, may provide important information when evaluating the T cell status in patients with chronic viral infections, such as HIV and hepatitis C virus (Gerlach et al., 1999
; Wasik et al., 2000
; Kostense et al., 2001
; Gruener et al., 2001
; Champagne et al., 2001
).
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Acknowledgments |
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References |
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Received 27 February 2002;
accepted 8 April 2002.