High numbers of IL-2-producing CD8+ T cells during viral infection: correlation with stable memory development

Nanna Ny Kristensen1, Jan Pravsgaard Christensen1 and Allan Randrup Thomsen1

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


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Using infections with lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus in mice as model systems, we have investigated the ability of antigen-primed CD8+ T cells generated in the context of viral infections to produce IL-2. Our results indicate that acute immunizing infection normally leads to generation of high numbers of IL-2-producing antigen-specific CD8+ T cells. By costaining for IL-2 and IFN-{gamma} intracellularly, we found that IL-2-producing cells predominantly constitute a subset of cells also producing IFN-{gamma}. Comparison of the kinetics of generation revealed that IL-2-producing cells appear slightly delayed compared with the majority of IFN-{gamma} producing cells, and the relative frequency of the IL-2-producing subset increases with transition into the memory phase. In contrast to acute immunizing infection, few IL-2-producing cells are generated during chronic LCMV infection. Furthermore, in MHC class II-deficient mice, which only transiently control LCMV infection, IL-2-producing CD8+ T cells are initially generated, but by 4 weeks after infection this subset has nearly disappeared. Eventually the capacity to produce IFN-{gamma} also becomes impaired, while cell numbers are maintained at a level similar to those in wild-type mice controlling the infection. Taken together, these findings indicate that phenotyping of T cell populations based on capacity to produce cytokines, and especially IL-2, can provide important information as to the functional status of the analysed cell subset. Specifically, combined analysis of the capacity to produce IL-2 and IFN-{gamma} can be used as a predictor for loss of function within the CD8+ T cell compartment.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Generally, CD4+ T cells are assumed to contribute to the clonal expansion and differentiation of CD8+ T cells in two distinct ways: (i) activated CD4+ T cells enhance the capacity of APCs (through upregulation of costimulatory molecules e.g. B7.1 and B7.2) to trigger naive CD8+ T cells (Bennett et al., 1998 ; Ridge et al., 1998 ; Schoenberger et al., 1998 ); and (ii) CD4+ T cells are believed to produce most of the IL-2 required for subsequent proliferation and differentiation (Cousens et al., 1995 ). However, quite often viral infection induces CD4+ T cell-independent primary CD8+ T cell responses (Buller et al., 1987 ; Moskophidis et al., 1987 ; Ahmed et al., 1988 ; Bodmer et al., 1993 ; Christensen et al., 1994 ), thus suggesting that both functions of CD4+ T cells may be redundant under certain circumstances. Because many viruses by themselves have the capacity to induce APC activation (Wu & Liu, 1994 ; Bachmann et al., 1998 ; Andreasen et al., 2000 ), it is perhaps not so difficult to understand why CD4+ T cells may be superfluous in this capacity during the inital stages of antiviral CD8+ T cell responses. But how is the requirement for IL-2 circumvented? One possibility is that other cytokines may substitute for IL-2, e.g. IL-15 (Kennedy et al., 2000 ). This is not the entire explanation, however, as IL-2-deficient mice are markedly impaired in their ability to raise antiviral CD8+ T cell responses (Cousens et al., 1995 ). Furthermore, a key role for IL-2 in effector-cell differentiation is supported by a recent study indicating that, while both IL-2 and IL-15 support proliferation and survival of activated CD8+ T cells in vitro, a high level of IL-2 is required for induction of an effector phenotype and function (Manjunath et al., 2001 ). Therefore, another possibility is that virus-activated CD8+ T cells themselves produce the IL-2 required for their initial clonal expansion and differentiation. A few reports have investigated the capacity of virus-activated CD8+ T cells to produce IL-2 (Kasaian & Biron, 1989 ; Mizuochi et al., 1989 ), and some evidence indicates that CD8+ T cells may transiently have this capacity. However, a thorough analysis applying recent quantitative approaches to cellular immunology has not been conducted. Using detection of peptide-induced (and thus antigen-specific) intracellular IL-2 to visualize and quantify cytokine-producing T cells, we have studied the capacity of CD8+ T cells to produce IL-2 in the context of an antiviral immune response. Our analysis indicates that IL-2-producing CD8+ T cells substantially outnumber IL-2-producing CD4+ T cells at all time points during acute viral infection (even though the frequency of these cells may be lower within the former subset). Furthermore, failure to generate and/or maintain IL-2-producing CD8+ T cells correlates with failing capacity to control virus levels in vivo, indicating that inability to produce this cytokine may serve as an early marker for functional defects within the CD8+ T cell subset.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Mice.
Female BALB/cA and C57BL/6 (B6) mice were purchased from M & B (Ry, Denmark). Female MHC class II-deficient (MHC class II -/-) mice and matched wild-type littermates, back-crossed five to six times on to a B6 background, were obtained from Taconic Farms (Germantown, NY, USA). Mice were always allowed to rest for at least 1 week before entering into experiments, at which time the animals were usually 7–9 weeks old. All animals were housed under controlled (specific pathogen-free) conditions that included testing of sentinels for unwanted infections according to the Federation of European Laboratory Animal Science Association standards; no such infections were detected.

{blacksquare} 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 ).

{blacksquare} 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.

{blacksquare} 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 {alpha}-chain of lymphocyte Peyer’s patch adhesion molecule-1 and very late Ag-4 (VLA-4)], FITC- and PE-conjugated anti-IFN-{gamma}, PE-conjugated anti-IL-2 and matched isotype controls.

{blacksquare} MHC/peptide tetramers for flow cytometry.
H-2Ld/np118–126, H-2Db/gp33–41 and H-2Db/np396–404 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.

{blacksquare} 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-{gamma} 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 gp33–41, np396–404 and np118–126) or 1 µg/ml (LCMV gp61–80 and VSV np52–59). After 5–6 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).


   Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Generation of high numbers of IL-2-producing CD8+ T cells during acute LCMV infection
Initial experiments were aimed at determining whether IL-2-producing CD8+ T cells are generated during acute infection with LCMV. Analysing the response to three class I-restricted viral peptides (gp33–41, np396–404 and np118–126), which constitute immunodominant epitopes in H-2b and H-2d mice, respectively (Oldstone et al., 1988 ; Schulz et al., 1989 ; Hudrisier et al., 1997 ), we consistently detected a small but distinct population of IL-2-producing CD8+ T cells in the spleens of mice infected 9–11 days earlier (for a representative result, see Fig. 1; a plot of IL-2-producing CD4+ T cells is included for comparison). Further analysis was therefore carried out to establish the kinetics of this response in virus-infected mice, comparing numbers of IL-2-producing cells with the number of IFN-{gamma}-producing CD8+ cells with the same peptide specificity. B6 mice were infected with LCMV Traub, and the number of gp33–41-specific, cytokine-producing CD8+ T cells was determined 7, 9, 11, 14 and 40 days after infection. For further comparison, we also evaluated the number of IFN-{gamma}- and IL-2-producing CD4+ cells specific for a known immunodominant MHC class II epitope (gp61–80) (Oxenius et al., 1995 ; Varga & Welsh, 2000 ). As can be seen from Fig. 2, the generation of IL-2-producing CD8+ T cells was slightly delayed relative to the generation of IFN-{gamma}-producing CD8+ T cells and, in relative terms (Fig. 2D), IL-2-producing cells became more frequent with transition into the memory phase (days 9–14 after infection). Thus, the ratio between IFN-{gamma}- and IL-2-producing CD8+ T cells initially was approximately 12:1, while the ratio was approximately 5:1 in memory mice analysed about 2 months after infection. Notably, the number of IFN-{gamma}-producing cells per IL-2-producing cell was significantly higher for CD8+ cells compared with CD4+ cells throughout the observation period. With regard to the relative prevalence of IL-2-producing CD4+ and CD8+ T cells, our results indicate that the frequency of IL-2-producing cells within each subset is roughly similar, perhaps with a bias for a higher frequency of IL-2-producing cells within the CD4+ T cell subset early in the infection (see also Fig. 1). However, due to the more extensive clonal expansion within the CD8+ subset (Fig. 2A), the absolute number of CD8+ T cells producing IL-2 in response to a single immunodominant epitope rapidly exceeded the number of IL-2-producing CD4+ T cells directed towards a comparable immunodominant class II epitope. [Since the frequency of IFN-{gamma}-producing cells relative to the fraction of phenotypically activated (VLA-4high) cells is of similar magnitude for the two selected epitopes (~25% and ~33% for CD4+ and CD8+ cells, respectively), we are inclined to believe that the responses to these epitopes are about equally representative for activated cells belonging to either T cell subset.]



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Fig. 1. IL-2 expression by CD4+ and CD8+ T cells during acute LCMV infection. B6 mice were infected intravenously with 200 p.f.u. of LCMV Traub, and on day 10 after infection cells were stimulated with gp33–41 (CD8+ T cells) or gp61–80 (CD4+ T cells) for 5 h. Following stimulation, cells were costained with anti-CD4 or anti-CD8 and anti-VLA-4, followed by staining for intracellular IL-2; staining with isotype-matched Ab was used as a control. The number in the upper right quadrant represents the percentage of VLA-4hiIL-2+ cells of the indicated T cell subset. Each plot, gated on CD4+ or CD8+ T cells, is representative of six animals analysed individually.

 


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Fig. 2. Kinetics analysis of IFN-{gamma} and IL-2 expression by CD4+ or CD8+ T cells during acute LCMV infection. Groups of B6 mice were infected with 200 p.f.u. of LCMV Traub and on the indicated days the total number of CD4+ and CD8+ T cells was determined (A). For each T cell subset, the total number of cells expressing intracellular IFN-{gamma} (B) or IL-2 (C) following peptide stimulation was also determined; peptides used were gp33–41 and gp61–80 for CD8+ and CD4+ T cells, respectively. In (D), the ratio of IFN-{gamma}- to IL-2-producing cells for each subset is shown. Bars represent the average of three animals ±SD. One of two identical experiments is depicted.

 
IL-2-producing CD8+ T cells constitute a distinct subset of cells also producing IFN-{gamma}
To establish the relationship between T cells producing the two cytokines, co-staining for IL-2 and IFN-{gamma} was carried out for gp33–41-specific CD8+ T cells taken from B6 mice 9, 11, 14 and 50 days after i.v. infection with LCMV Traub. This analysis (Fig. 3) revealed that, during the acute response, IL-2 was being produced primarily by a subset of CD8+ T cells also producing high levels of IFN-{gamma}, and a similar pattern was obtained after analysing the response to np396–404 in B6 mice and to np118–126 in BALB/c mice (data not shown). Compared with virus-specific CD4+ T cells, the initial frequency of virus-specific CD8+ T cells producing both cytokines relative to the frequency of cells producing IFN-{gamma} only was substantially lower (Fig. 3), and with time the relative frequency of coproducing cells increased for CD8+ T cells. The same pattern was seen for CD4+ T cells, in which case most memory cells producing IFN-{gamma} also produced IL-2.



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Fig. 3. Coexpression of cytokines during acute LCMV infection. B6 mice were infected with 200 p.f.u. of LCMV Traub and on the indicated days coexpression of IFN-{gamma} and IL-2 was determined in CD8+ (A) or CD4+ (B) T cells; peptides used were gp33–41and gp61–80 for CD8+ and CD4+ T cells, respectively. Numbers refer to the percentage of CD8+ or CD4+ T cells that are IFN-{gamma}+IL-2+ (top) and IFN-{gamma}+IL-2- (bottom); the frequency of cells producing IL-2 only was generally <=0·5%. Each plot, gated on CD4+ or CD8+ T cells, is representative of at least six animals analysed individually.

 
Generation of IL-2-producing CD8+ T cells in VSV infected mice
To see if other viral infections also induced antigen-specific CD8+ T cells with the capacity to produce IL-2, mice were infected with VSV. At the peak of the primary response [day 7 post-infection (p.i.)] (Andreasen et al., 2000 ) and 2 weeks later, CD8+ T cells were evaluated for their capacity to produce IL-2 and IFN-{gamma} when stimulated with the immunodominant epitope np52–59. As can be seen in Fig. 4, CD8+ T cells producing IL-2 were also generated during VSV infection and, similar to the situation in LCMV-infected mice, most IL-2-producing cells constituted a subset of the IFN-{gamma}+ population.



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Fig. 4. Cytokine expression during acute VSV infection. B6 mice were infected with 106 p.f.u. of VSV Indiana. (A) On day 7 after infection, splenocytes were stimulated with VSV np52–59 and then costained with anti-CD8, anti-VLA-4 and anti-IFN-{gamma} or anti-IL-2; numbers refer to the percentage of CD8+ T cells positive for cytokine expression. (B) On day 7 and 21 after infection, peptide-stimulated cells from the spleen and peritoneum were costained with anti-CD8, anti-IFN-{gamma} and anti-IL-2; numbers refer to the percentage of CD8+ T cells that are IFN-{gamma}+IL-2+ (top) and IFN-{gamma}+IL-2- (bottom). Each plot, gated on CD8+ T cells, is representative of five to eight animals analysed individually.

 
Parallel to the analysis of splenic CD8+ T cells, we also analysed CD8+ T cells harvested from the peritoneum. This was done to investigate whether any difference in cytokine profile existed between cells present in secondary lymphoid tissue (spleen) and effector cells that have migrated to a non-lymphoid organ site (peritoneum) (Masopust et al., 2001 ). Both during the acute response and in immune mice, it was found that a much higher frequency of antigen-specific CD8+ T cells present in the peritoneum were producing both IL-2 and IFN-{gamma} compared with splenic CD8+ T cells taken at the same time. Since there is no evidence for local replication of VSV in the peritoneum following i.v. infection, this pattern suggests that coproducing cells are preferentially represented among mature effector cells leaving the secondary lymphoid tissues.

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-{gamma} 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 gp33–41 and np396–404 were studied; however, since essentially similar patterns were obtained, only data for gp33–41 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-{gamma}-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-{gamma} (albeit at a reduced level, see Fig. 5). Here the difference in the ratio of IFN-{gamma}+ to tetramer+ cells in the two groups should be noted: because the gp33–41 peptide contains both a Db- and a Kb-restricted immunodominant epitope (Hudrisier et al., 1997 ), more IFN-{gamma}+ 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-{gamma} 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-{gamma} 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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Table 1. Comparison of cytokine expresssion following infection with Armstrong or clone 13 LCMV

 


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Fig. 5. Cytokine expression by CD8+ T cells from LCMV-infected animals varies with type of infection. Groups of B6 animals were infected with either 5x103 p.f.u. of LCMV Armstrong (upper panel) or 106 p.f.u. of LCMV Clone 13 (lower panel). On day 10 after infection splenocytes were stimulated with gp33–41 for 5 h. Following stimulation, cells were costained with anti-CD8, anti-VLA-4 and H-2Db/gp33–41 tetramer (tet) or anti-IFN-{gamma} or anti-IL-2; in addition costaining with anti-CD8, anti-IFN-{gamma} and anti-IL-2 was performed. Numbers refer to the percentage of CD8+ T cells that are positive for the indicated markers. Each plot, gated on CD8+ T cells, is representative of four animals analysed individually. At this time point, Armstrong-infected mice have cleared the infection while clone 13-infected mice contain high levels of virus (spleen: 3·5x107 p.f.u./g, lungs: 5x107 p.f.u./g; mean of four mice).

 
CD4+ T cells are not required for generation of IL-2-producing CD8+ T cells, but are pivotal for their maintenance
From previous studies, it is known that in LCMV Traub-infected MHC class II -/- mice, the virus infection is initially controlled at a low level, but between 1 and 3 months after infection, infectious virus reappears in the blood (Thomsen et al., 1996 ). Furthermore, at this time point, CD8+ T cells are virtually unable to differentiate into secondary cytotoxic effectors in vivo (Thomsen et al., 1996 ). To study how this is reflected in the phenotype of antigen-specific CD8+ T cells present in CD4+-deficient mice, mice deficient in MHC class II expression were infected with LCMV Traub, and numbers of IL-2- and IFN-{gamma}-producing CD8+ T cells, as well as numbers of tetramer+ cells, were compared with those in matched wild-type mice. As expected from the analysis of virus levels (Thomsen et al., 1996 ), we found that class II -/- mice initially generated an almost unimpaired CD8+ T cell response evaluated in terms of IFN-{gamma}- and IL-2-producing cells (Fig. 6). However, by 4 weeks after infection (i.e. before virus has reappeared in the blood; Thomsen et al., 1996 ), few IL-2-producing CD8+ T cells could be detected in MHC class II -/- mice (see Fig. 7A for representative plots), while less than a twofold difference was found regarding numbers of tetramer+ and IFN-{gamma}-producing CD8+ T cells (Figs 6 and 7). Thus, the ratio between IL-2+ and IFN-{gamma}+ CD8+ T cells was 0·05–0·08 in class II -/- mice and 0·17–0·21 in wild-type controls (ranges for four mice/group); this difference in ratios was statistically significant (P<0·05, Mann–Whitney rank sum test). By day 90 p.i., the frequency of cells staining for IL-2 in knockout mice did not exceed the level for the isotype control. Furthermore, the signal for IFN-{gamma} (evaluated in terms of mean fluorescence intensity) in CD8+ T cells from these mice was consistently decreased compared with wild-types (six mice/group were analysed; see Fig. 7A for representative histograms) and in contrast to wild-type mice, class II -/- mice had clearly detectable levels of virus in their organs (e.g. lungs: 7·5x104 p.f.u./g, mean of six mice). In contrast to the results of the functional analysis, we detected no difference between knockout mice and wild-type controls in the number of virus-specific (tetramer+) cells present in the spleen (Fig. 6), nor did we find evidence for T cell receptor downmodulation as judged from the intensity of tetramer staining (Fig. 7B). Thus, virus-primed CD8+ T cells gradually lose functional capacity in virus-infected MHC class II -/- mice lacking a normal CD4+ T cell subset and, notably, the capacity to produce IL-2 is lost before the capacity to synthesize IFN-{gamma}.



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Fig. 6. Kinetic analysis of cytokine expression by CD8+ T cells from MHC class II -/- mice during LCMV infection. Groups of MHC class II -/- and wild-type control mice were infected with 200 p.f.u. of LCMV Traub. (A) On days 7, 9 and 11 after infection, the total number of CD8+ T cells and the total number of gp33–41-stimulated CD8+ T cells producing IFN-{gamma} or IL-2 was determined. (B) On day 28 and 90 after infection, the total number of gp33–41-specific CD8+ T cells that were tetramer+ (tet+), IFN-{gamma}+ or IL-2+ was determined. Bars represent the average of three to six animals±SD.

 


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Fig. 7. Phenotype of virus-specific CD8+ T cells from LCMV-infected MHC class II -/- mice as a function of time. Groups of MHC class II -/- and wild-type control mice were infected with 200 p.f.u. of LCMV Traub. (A) On days 11, 28 and 90 after infection, gp33–41-stimulated CD8+ T cells were stained with anti-IFN-{gamma} and anti-IL-2. Left, representative dot plots, gated on CD8+ T cells. Numbers refer to the percentage of CD8+ T cells that are IFN-{gamma}+IL-2+ (top) and IFN-{gamma}+IL-2- (bottom). Right, histograms showing IFN-{gamma} expression by activated (VLA-4hi) CD8+ T cells on the indicated days. Numbers refer to the percentage of CD8+ T cells that are IFN-{gamma}+ positive. (B) Ninety days after infection, splenocytes were stained using a combination of anti-CD8, anti-VLA-4 and H-2Db/gp33–41 tetramers. Gates were set for CD8+ T cells. All findings are representative of four to six mice analysed individually.

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
T cell responses to viral infections are typically characterized by massive CD8+ T cell expansion and differentiation (Butz & Bevan, 1998 ; Murali-Krishna et al., 1998 ). In many cases, this CD8+ T cell activation can take place independently of CD4+ T cells, at least during the primary response (Buller et al., 1987 ; Moskophidis et al., 1987 ; Ahmed et al., 1988 ; Bodmer et al., 1993 ; Christensen et al., 1994 ). In contrast, IL-2-deficient mice appear to be severely immunocompromised (Cousens et al., 1995 ), which is in total agreement with recent in vitro findings suggesting that IL-2 is pivotal for CD8+ effector T cell differentiation (Manjunath et al., 2001 ). Together, these findings point to the likelihood that IL-2 can be produced in sufficient amounts by cell types other than CD4+ T cells.

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 10–20% 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-{gamma}, 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-{alpha} and IFN-{gamma}, 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-{gamma} 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-{gamma}. Since IFN-{gamma} 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-{gamma} 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-{gamma} production is found in late-stage patients (Lieberman et al., 2001 ).

An important issue not resolved by this study is the cause–effect 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-{gamma} 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-{gamma} 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 ).


   Acknowledgments
 
This study was supported in part by the Danish Medical Research Council, the Biotechnology Center for Cellular Communication and the Novo Nordisk Foundation. J.P.C. is the recipient of a Research Fellowship from the Weimann Foundation, University of Copenhagen, Denmark.


   References
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Abstract
Introduction
Methods
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Discussion
References
 
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Received 27 February 2002; accepted 8 April 2002.