Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark
Correspondence
Jan Pravsgaard Christensen
j.pravsgaard{at}immi.ku.dk
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ABSTRACT |
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INTRODUCTION |
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Despite the fact that it is becoming increasingly evident that CD8+ T cells may be relevant as producers of cytokines, there are still a number of questions that need to be addressed. What is the range of cytokines at the population level as well as at the level of the individual cell which may be produced by in vivo-primed antigen-specific CD8+ T cells? Is this profile fixed or does it change as a function of time and the state of activation (effectormemory cell)? How do cytokine-producing CD8+ T cells compare qualitatively and quantitatively to antigen-specific CD4 T cells primed in parallel? The primary aim of this study was therefore to determine the ability of virus-specific CD8+ T cells to produce cytokines as a function of their activation state, and to define some of the parameters influencing the spectrum of cytokines produced. Cells were analysed ex vivo using primarily intracellular staining for cytokines and flow cytometry. This type of analysis makes it possible not only to determine the spectrum of cytokines, which can be produced at the population level, but also allows a precise evaluation of the number of cells involved as well as the relationship between subset of cells producing different cytokines.
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METHODS |
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Viruses and virus infections.
The viscerotropic Traub strain of LCMV was administered i.v. or i.p. in a dose of approx. 200 p.f.u.; inoculation by these routes results in non-lethal, immunizing infection (Nansen et al., 1999). VSV of the Indiana strain was used in a dose of 106 p.f.u. i.v. This dose is non-lethal in immunocompetent mice, but induces a potent CD8+ T cell response (Andreasen et al., 2000
). For co-infection experiments (VSV and LCMV) the less invasive LCMV strain (Armstrong clone 53b) was used; this strain was used in a dose of 5000 p.f.u. i.v. All viruses were produced, stored and quantified as described before (Marker & Volkert, 1973
; Thomsen et al., 1997
).
Cell preparation.
Spleens were removed from mice killed by cervical dislocation. Single-cell suspensions of spleen cells were obtained by pressing the organs through a fine steel mesh. Peritoneal cells were obtained by lavage with 5 ml of ice-cold Hanks' balanced salts solution.
Adoptive transfer of TCR transgenic CD8+ T cells.
Single-cell suspensions of spleen cells from TCR-318 or TgN(N15) mice were obtained as described above, and 13x106 splenocytes were transferred i.v. to B6 mice. TCR+CD8+ T cells from TCR-318 donor mice were tracked using a combination of anti-V2 and -V
8 antibodies; <2 % of wild-type cells express this combination, and transgenic cells were only analysed functionally when
20 % of splenic CD8+ T cells expressed this receptor combination. Since no Ab was commercially available for the V
chain expressed by TCR+CD8+ T cells from TgN(N15) donor mice, these cells were tracked using anti-V
8 only. However, V
8+ cells specific for np5259 comprise <1 % of the CD8+ T cells in untransplanted mice, while only recipients in which np5259 specific V
8+ cells had expanded to >8 % of splenic CD8+ T cells were accepted for analysis of donor cell cytokine phenotype. Thus, in both cases contamination with recipient cells represented
10 % or less of the cells analysed.
Monoclonal antibodies for flow cytometry.
The following mAbs were purchased from BD PharMingen as rat anti-mouse mAbs: CyChrome (Cy)-conjugated anti-CD4 and anti-CD8a, FITC-conjugated anti-CD49d [common -chain of lymphocyte Peyer's patch adhesion molecule-1 and very late Ag-4 (VLA-4)], FITC-conjugated anti-CD44, PE-conjugated anti-V
8, FITC-conjugated anti-V
2, FITC-conjugated anti-V
8, FITC- and PE-conjugated anti-IFN-
, PE-conjugated anti-IL-3, anti-IL-4, anti-IL-5, anti-IL-10, anti-GM-CSF, PE- and APC-conjugated anti-IL-2, PE- and FITC-conjugated anti-TNF-
and matched isotype controls.
MHC/peptide tetramers (tet) for flow cytometry.
H-2Kb/np5259 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.
To detect intracellular cytokines, splenocytes were cultured for 5 h at 37 °C in complete RPMI 1640 medium supplemented with murine recombinant IL-2 (50 U ml1), monensin (3 µM) and relevant peptides at a concentration of 0·1 µg ml1 (LCMV gp3341 and np118126) or 1 µg ml1 (LCMV gp6180 and VSV np5259). After stimulation the cells were stained with relevant antibodies as described previously (Kristensen et al., 2002). For tetramer staining, cells were incubated with tetramers for 30 min at 4 °C, at which time mAbs for surface labelling were added; the cells were then incubated for a further 30 min at 4 °C, before washing and fixation (Christensen et al., 2001
). Sample data were acquired using a FACSCalibur (BD Biosciences), and results were analysed using CellQuest software (BD Biosciences). Isotype control mAbs and in some cases also unstimulated cells were included to define appropriate cut-off levels.
Quantification of chemokine production.
Supernatants harvested from peptide (1 µg ml1) and unstimulated cultures of splenic cells incubated for 6 h in vitro were assayed for chemotactic cytokines using commercially available sandwich ELISA kits (R&D systems).
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RESULTS |
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As shown previously (Butz & Bevan, 1998; Murali-Krishna et al., 1998
; Slifka & Whitton, 2000
; Varga & Welsh, 2000
; Kristensen et al., 2002
), primary virus-specific CD4+ and CD8+ effector T cells produce the cytokines IFN-
, TNF-
and IL-2 (Fig. 1
A). A substantial subset of primary effector T cells also produced GM-CSF. Besides producing proinflammatory cytokines, MHC class I-restricted T cells were also found to be the predominant source of the chemotactic cytokines RANTES, MIP (macrophage inflammatory protein)-1
and
(Fig. 2
). In contrast, T cells producing type 2 cytokines (IL-4, IL-5 and IL-10) or IL-3 were either absent or found at marginal frequencies within both T cell compartments (Fig. 1B and C
and data not shown).
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It should be noted that although both virus-specific CD4+ and CD8+ T cells are clearly type 1 cells, the distribution of cytokine-producing subsets differs. Similar numbers of CD4+ T cells produce TNF-, IL-2 or GM-CSF during the primary response whereas TNF-
-producing cells are more prevalent amongst CD8+ effector T cells. Following contraction of the T cell population, equal numbers of IFN-
-, TNF-
- and IL-2-producing CD4+ T cells were observed (Fig. 1C
). As with CD8+ T cells, GM-CSF-producing cells were less prevalent in the memory phase.
Virus-specific CD8+ T cells producing TNF-, GM-CSF or IL-2 constitute overlapping subpopulations of cells also producing IFN-
To study the relationship between CD8+ T cells producing different cytokines, gp3341 stimulated CD8+ effector T cells were co-stained for two of the following cytokines: IFN-, TNF-
, GM-CSF and IL-2 (Fig. 3
).
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Cytokine profile of virus-specific CD8+ T cells generated during primary VSV infection
To investigate whether the same cytokines would be produced by virus-specific CD8+ T cells generated in response to another virus, B6 mice were inoculated i.v. with VSV. Infectious VSV can only be isolated briefly from the spleen, and this CD8 T cell response therefore represents programmed expansion and differentiation of cells in the absence of actual infection. On day 7 (the peak of the primary response) and day 21 (memory state) after infection, we analysed the capacity of np5259 specific (immunodominant epitope in H-2b mice) CD8+ T cells to produce cytokines (Fig. 4).
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Cytokine phenotype of virus-specific CD8+ T cells as a function of localization
Besides splenic T cells, we evaluated the ability of LCMV- and VSV-specific CD8+ T cells from the peritoneum to produce cytokines (Table 1 and 2). The peritoneum represents a tertiary organ site, and the cells present here are likely to represent a more differentiated phenotype compared with cells obtained from secondary lymphoid tissues (Masopust et al., 2001
).
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To investigate whether such signals were essential, we analysed virus-specific cells harvested from the peritoneum of mice infected with VSV i.v.; in this case no inflammatory response is observed (no increase in cell numbers).
At the height of the primary response to VSV, not only the frequency of IFN- producers (Table 2
), but also the level of per-cell expression was significantly higher for peritoneal cells than for splenocytes (data not shown). In addition, the ratio of cytokine+/tetramer (tet)+ cells was significantly higher in peritoneum, and, as in LCMV-infected mice, a higher frequency of peritoneal CD8+ T cells produced more than one cytokine (all TNF-
-producing cells also produced IFN-
) (Table 2
). Twenty one days after infection the majority of VSV-specific (tet+) CD8+ T cells both in the spleen and in the peritoneum produced IFN-
. About half the virus-specific CD8+ T cells in the spleen were also TNF-
+ whereas nearly all virus-specific CD8+ T cells harvested from the peritoneum co-produced TNF-
. Thus, the proportion of co-producing cells increased with time in both spleen and peritoneum. Moreover, cells with this phenotype were found at a higher frequency in the peritoneum independently of local inflammatory signals (Table 2
).
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Naive B6 mice were given spleen cells from TCR transgenic mice (TCR-318) expressing a V2/V
8·2 T cell receptor specific for LCMV gp3341. The day after cell transfer, recipients were infected with LCMV, and 9 days after infection TCR+ CD8+ T cells were analysed for the ability to produce cytokines (Fig. 6
). Despite the uniform expression of a single clonotype, in vivo activated TCR+ cells not only produced the same spectrum of cytokines (IFN-
, TNF-
, IL-2 and GM-CSF) (Fig. 6
) as did gp3341-specific cells from normal mice (see Fig. 1
for comparison), but the distribution of cytokine-producing cells also matched that observed for polyclonal cells with the same specificity.
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IFN- and IL-12 is not required to inhibit the generation of TC2 cells during primary VSV infection
From in vitro studies it is known that CD8+ T cells cultured with antigen in the presence of anti-IFN- (and IL-4) will develop a TH2 like phenotype (TC2 cells) (Croft et al., 1994
; Sad et al., 1995
). To see if TC2 cells would be generated in vivo in the absence of IFN-
, IFN-
/ mice were infected with VSV (failure to the produce IFN-
does not influence the course of VSV infection) (Andersen et al., 1999
), and on day 7 p.i. we looked for np5259 specific cells producing typical TC2 cytokines (IL-4, IL-5 and IL-10) as well as TNF-
(positive control). No VSV-specific CD8+ T cells from IFN-
/ mice (n=3) produced typical TC2 cytokines (not shown) indicating that IFN-
is not required to block TC2 differentiation in vivo. It should be stressed that absence of IFN-
does not impair the generation of VSV-specific cells since (i) the generation of TNF-
-producing cells was unimpaired and (ii) other experiments revealed that the number of splenic tet+ CD8+ cells was similar in IFN-
/ and wild-type mice (data not shown). Similar to the findings in IFN-
/ mice absence of IL-12
did not influence the cytokine profile (data not shown).
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DISCUSSION |
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CD8+ T cells can be divided into TC1 and TC2 subsets producing the same range of cytokines as the corresponding TH subsets (Croft et al., 1994; Maggi et al., 1994
; Le Gros & Erard, 1994
; Sad et al., 1995
). However, virus-specific CD8+ T cells seem to have a strong propensity to develop a TC1 cytokine profile: IL-4-, IL-5- and IL-10-producing cells were not found in either LCMV-infected B6 or BALB/c (data not shown) mice or following VSV infection. IFN-
and IL-12 is known to regulate the in vitro differentiation of CD8+ T cells into a type 1 cytokine-producing phenotype. However, similar to the situation for TH cells (Schijns et al., 1994
; Oxenius et al., 1999
), we did not find type 2 TC cells in IFN-
or IL-12
deficient mice. Notably, this type 1 profile was observed even following inoculation of a virus (VSV), which undergo very limited replication in the host (no infectious VSV can be detected in spleen beyond day 2 p.i.). Taken together these observations suggest that CD8+ T cells intrinsically are hardwired towards type 1 differentiation. Consistent with this assumption, and unlike the situation for CD4+ T cells, very few instances of in vivo-generated type 2 CD8+ T cells have been described (Salgame et al., 1991
; Maggi et al., 1994
).
Virtually all virus-specific CD8+ T cells generated during primary LCMV infection produce IFN- (Butz & Bevan, 1998
; Murali-Krishna et al., 1998
). Part of these cells also produce TNF-
and IL-2 (Slifka & Whitton, 2000
; Kristensen et al., 2002
). Here, we extend these observations by showing that a subpopulation of IFN-
-producing CD8+ T cells may also produce GM-CSF. More important, we find that although virus-specific CD8+ T cells are quite heterogeneous in their ability to produce cytokines, the ability to produce more than one cytokine is not a random occurrence. Indeed there is a significant trend for co-production of several cytokines. The frequency of co-producing cells was significantly higher in the peritoneum than in the spleen independently of local inflammatory signals. This may indicate that co-producing CD8+ T cells are to be found mainly among fully differentiated cells with the potential to leave the secondary lymphoid organs and migrate into tertiary tissues. Notably, there also seems to be a preferential survival of these multipurpose cells as the relative frequency of this phenotype increases following contraction of the CD8+ T cell population. These observations expand on existing data (Slifka & Whitton, 2000
; Kristensen et al., 2002
) suggesting that there is a preferential maintenance of a T cell phenotype characterized by the potential to produce several cytokines. Interestingly, the ability to produce all cytokines is not maintained; LCMV-specific CD8+ T cells producing GM-CSF are only abundant during the effector phase and preferentially disappear with transition into the memory phase.
Interestingly, few GM-CSF-producing cells are generated during VSV infection, and overall VSV-specific CD8+ T cells appear to be less functionally differentiated during the primary response than do LCMV-specific cells: while the ratio of IFN-+/tet+ is close to one for LCMV at the time of peak effector cell numbers (Butz & Bevan, 1998
; Murali-Krishna et al., 1998
), this ratio is only about 1 : 2 in VSV infected mice (present report). Furthermore, per-cell IFN-
production is lower [lower mean fluorescence intensity (MFI)], and fewer VSV-specific, IFN-
+ CD8+ T cells co-produce TNF-
. With time these differences tend to diminish. One explanation for this pattern could be that during acute VSV infection, many antigen-specific CD8+ T cells are generated, which are arrested at an early stage of differentiation and therefore preferentially lost during the contraction phase. Part of the reason why functional differentiation may be less complete following VSV infection as compared with LCMV infection, could be a difference in the state of the APCs during the two infections (Ruedl et al., 1999
). Consistent with this assumption we find that numbers and per-cell capacity of VSV-specific cells to produce IFN-
and TNF-
increase in co-infected animals. However, no induction of GM-CSF-producing cells was observed, indicating that additional factors influence cytokine production perhaps the transient nature of antigen presentation in VSV infected mice (Andreasen et al., 2000
; Christensen et al., 2002
) also plays a role. Supporting that the environment in which antigen presentation takes place may influence the overall cytokine profile, we found that naive TCR+ cells specific for immunodominant epitopes of either virus differentiate into cytokine-expression patterns which reproduce the differences seen for polyclonal cells of the same specificity (cf. production of TNF-
and GM-CSF).
From other studies it appears that different thresholds exist for induction of different cytokines (Valitutti et al., 1996; Itoh & Germain, 1997
). Thus, a likely assumption would be that much of the heterogeneity in cytokine production by cells with the same epitope specificity reflected the normal diversity of the involved TCRs, i.e. cells with high avidity for their antigen might be activated to produce a wider range of cytokines than low avidity cells. However, in vivo-primed TCR transgenic cells were as heterogeneous in their cytokine profile as were a normal polyclonal T cell population with the same epitope specificity. Thus, a diverse avidity spectrum does not suffice to explain the heterogeneity in cytokine phenotypes. This conclusion is consistent with previous findings demonstrating intraclonal heterogeneity in cytokine production by long-term CD4+ T cells in culture (Itoh & Germain, 1997
).
In conclusion, our results underscore the extensive range of cytokines that may be produced by in vivo activated CD8+ T cells. Interestingly, almost similar cytokine profiles are generated whether or not extensive virus replication takes place. This may suggest a deeply founded propensity of CD8+ T cells for type 1 differentiation, which may be linked to their evolutionary role as effector cells controlling parasites with a cytoplasmic habitat.
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ACKNOWLEDGEMENTS |
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Received 15 December 2003;
accepted 6 February 2004.