Virus-induced non-specific signals cause cell cycle progression of primed CD8+ T cells but do not induce cell differentiation

Susanne Ørding Andreasen, Jan Pravsgaard Christensen1, Ole Marker and Allan Randrup Thomsen

Institute of Medical Microbiology and Immunology, University of Copenhagen, The Panum Institute, 3C Blegdamsvej 2200, Copenhagen N, Denmark

Correspondence to: A. Randrup Thomsen


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this report the significance of virus-induced non-specific T cell activation was re-evaluated using transgenic mice in which about half of the CD8+ T cells expressed a TCR specific for amino acids 33–41 of lymphocytic choriomeningitis virus glycoprotein I. This allowed tracing of cells with known specificity and priming history in an environment also containing a normal heterogeneous CD8+ population which served as an intrinsic control. Three parameters of T cell activation were analyzed: cell cycle progression, phenotypic conversion and cytolytic activity. Following injection of the IFN inducer poly(I:C), proliferation of memory (CD44hi) CD8+ T cells but no phenotypic or functional activation was observed. Following injection of an unrelated virus [vesicular stomatitis virus (VSV)], naive TCR transgenic cells did not become significantly activated with respect to any of the parameters investigated. In contrast, memory TCR transgenic cells were found to proliferate extensively early after VSV infection (day 0–3), whereas limited proliferation was observed later (day 3–6) when proliferation of non-transgenic CD8+ T cells is maximal. This aborted response did not result from anergy to TCR stimulation, as memory TCR transgenic cells proliferated vigorously upon stimulation with their nominal peptide. Despite the massive proliferation of memory cells observed early after VSV infection, no phenotypic or functional activation was observed. Together these findings indicate that both non-specific and antigen-specific signals contribute to the initial virus-induced proliferation of CD8+ T cells, but for further proliferation and differentiation to take place, TCR–ligand interaction is required. The implications for maintenance of T cell memory is discussed.

Keywords: bystander activation, cytokines, T cell memory, T cell turnover, viral infection


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Many viral infections induce massive expansion of the CD8+ T cell population and the majority of the cells generated also acquire an activated phenotype (VLA-4hiLFA-1hiCD44hiL-selectinlo) (17). Staining for intracellular IFN-{gamma} following ligation of the TCR with {alpha}CD3 has revealed that many of these CD8+ T cells are also functionally differentiated (8), but the antigenic specificity of most of these cells eluded analysis until very recently. Initial experiments trying to determine the frequency of virus-specific CD8+ T cells were based on limiting dilution assays. With this technique 1–10% of the CD8+ T cells were found to be virus specific (4,5,9). As specificity is one of the hallmarks of the adaptive immune system, this low frequency of virus-specific CD8+ T cells was rather surprising and led researchers in this field to introduce a new concept: bystander activation. The mechanisms underlying this seemingly non-specific activation of T cells were poorly characterized, but two hypotheses prevailed—either activation was due to some degree of cross-reactivity at the level of the TCR (10,11) or it was driven by cytokines released in the inflammatory environment generated in the context of most viral infections (12,13). The latter hypothesis was supported by in vitro experiments performed by Ehl et al. who showed that different cytokines (e.g. IL-2) were able to stimulate CD8+ T cells to become cytolytically activated (14). Furthermore, Tough et al. showed that in vivo injection of poly(I:C), which is a potent inducer of IFN-{alpha}/ß, lead to massive proliferation of the CD8+CD44hi cells (1).

Recently, two new methodological approaches have become available to directly quantify virus-specific T cells, i.e. staining with MHC–peptide tetramer complexes and evaluation of peptide-specific IFN-{gamma} synthesis either by flow cytometry or in ELISPOT assays (1518). Experiments applying these two techniques have recently revealed that in mice infected with lymphocytic choriomeningitis virus (LCMV) as much as 50–70% of the activated CD8+ T cells are virus-specific (17,18). This finding questioned the whole concept of bystander activation. However, it is still not possible to account for all activated CD8+ T cells. Furthermore, the situation regarding the ratio of virus-specific versus non-specific T cells may be different in other viral infections.

Consequently, the purpose of this study was to re-evaluate the significance of virus-induced non-specific T cell activation. This was done using mice with a high frequency of CD8+ T cells carrying a transgenic encoded TCR. This approach made it possible to follow the fate of both naive and primed CD8+ T cells during a viral infection for which they had no specificity. The mouse strain used was a transgenic line in which 50% of the CD8+ T cells express a TCR specific for amino acids 33–41 of the LCMV glycoprotein I (19). An additional advantage of using this particular transgenic line was that this frequency of T cells carrying the transgenic receptor allowed naturally heterogeneous T cells and T cells with known specificity to be directly compared following activation in the same environment. As a non-related virus we chose vesicular stomatitis virus (VSV), because it is known that no cross-reactivity exists between LCMV and VSV (20), and, moreover, VSV is known to induce substantial activation of CD8+ T cells (1). Three parameters of T cell activation were evaluated (21): cell cycle progression (in vivo incorporation of BrdU), phenotypic conversion (up-regulation of VLA-4 and down-regulation of L-selectin) and effector capacity (cytolytic activity).

Our analyses reveal that two separate components contribute to the marked proliferation of CD8+ T cells generally associated with viral infections: in the early phase of infection already primed CD8+ T cells (CD44hi = memory cells) proliferate extensively in response to non-specific stimuli, e.g. IFN-{alpha}/ß. However, this response is transient and for proliferation to continue at least some degree of antigen-specific triggering through the TCR is required. Notably, although the non-specific proliferation of memory cells does not lead to generation of effector T cells, this response may be crucial to sustain a sizable memory pool.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Female C57BL/6 were purchased from Bomholtgaard (Ry, Denmark). Transgenic mice expressing a V{alpha}2/Vß8.2 TCR specific for amino acids 33–41 of the LCMV glycoprotein I in association with H-2Db on 50% of CD8+ T cells (19) were bred locally from breeding pairs kindly provided by H. Pircher and R. M. Zinkernagel (Zürich, Switzerland). Type I IFN receptor knockout mice (IFN-{alpha}/ßR–/–, A129) were the progeny of breeding pairs obtained from B & K Universal (Hull, UK). C57BL/6CrlBR used as recipients for adoptive transfer were purchased from Charles River (Hannover, Germany). Mice from outside sources were always allowed to rest for 1 week before entering into experiments; by that time the animals were ~7–8 weeks old. Animals were housed under controlled conditions that included testing of sentinels for unwanted infections according to FELASA standards; no such infections were detected.

Virus infection
LCMV of the Traub strain was produced, stored and quantified as previously described (22). Mice to be infected with LCMV received 103 LD50 in an i.v. injection of 0.3 ml. Inoculation by this route is followed by transient, immunizing infection (23,24). When VSV of the Indiana strain (originally provided by K. Berg of this institute) was used, the virus dose was 106 p.f.u. This virus dose is non-lethal to immunocompetent mice, but induced a potent CD8+ T cell response (25). VSV was produced, stored and quantified as described previously (25).

In vivo peptide stimulation
The LCMV GP33–41 peptide was kindly provided by S. Buus of this institute. Mice were injected i.p. with 100 µg peptide dissolved in 200 µl of PBS.

Treatment with polyinosinic–polycytidylic acid [poly(I:C)]
The mice were injected i.p. with 150 µg poly(I:C) (Sigma, St Louis, MO) dissolved in 150 µl of PBS.

In vivo generation of LCMV-specific memory T cells
Single-cell suspensions were obtained by pressing the spleens from TCR transgenic mice through a fine steel mesh. The cells were washed 3 times, resuspended in HBSS and injected i.v. (2x106 cells/recipient) into C57BL/6CrlBR mice. Mice were infected with 103 LD50 of LCMV i.v. 2–3 days later (26).

In vivo BrdU labeling
Mice were given BrdU (Sigma) at 0.8 mg/ml in their drinking water for a period of 3 days (1). BrdU-containing water was protected from light and changed daily.

mAb
The following mAb were purchased from PharMingen (San Diego, CA) as rat anti-mouse antibody: FITC-conjugated anti-CD49d (common {alpha}4 chain of LPAM-1 and VLA-4) (R1-2), FITC-, phycoerythrin (PE)- and CyChrome-conjugated anti-CD8a (Ly-2) (53-6.7), biotinylated anti-L-selectin (CD62L) (MEL-14) (LECAM-1, Ly-22), FITC-conjugated anti-Vß8.1,8.2 TCR (MR5-2), PE-conjugated anti-V{alpha}2 TCR (B20.1), PE-conjugated anti-CD44 (Pgp-1, Ly-24, H-CAM), PE-conjugated anti-IFN-{gamma} and biotinylated anti-CD25 (IL-2 receptor {alpha} chain). For BrdU staining, FITC-conjugated anti-BrdU (Becton Dickinson, San Jose, CA) was used.

Fluorescence staining and flow cytometric analysis
Staining for flow cytometry was done as described previously (2,27). Briefly, 1x106 cells were incubated for 5 min in FACS medium (PBS containing 10% rat serum, 1% BSA and 0.1% NaN3). Subsequently, cells were incubated with relevant antibodies in the dark for 20 min at 4°C, after which they were washed 3 times in PBS with 0.1% NaN3 and fixed with 1% paraformaldehyde in PBS. In the case of biotin-conjugated antibody, cells were additionally incubated with streptavidin–TriColor (Caltag, Burlingame, CA) for 20 min before fixation.

For BrdU staining, cells were stained for surface markers as described above, resuspended in PBS + 1% NaN3, transferred to cold 0.15 M NaCl solution and fixed by adding cold 96% ethanol drop by drop. After a 30 min incubation on ice, cells were washed once with PBS and resuspended in PBS/0.01% Tween 20 and 1% paraformaldehyde. After a 1 h incubation at room temperature, cells were pelleted and resuspended in PBS, 0.15 M NaCl, 4.2 mM MgCl2, pH 5 containing 50 Kunitz units/ml DNase I (Sigma, St Louis, MO). After incubation for 15 min at 37°C, cells were washed once in PBS before adding the anti-BrdU antibody. After a 30 min incubation at room temperature, cells were washed in PBS and analyzed.

To detect intracellular IFN-{gamma}, splenocytes were cultured at 37°C in 96-well round-bottomed plates at a concentration of 1x106 cells/well in a volume of 0.2 ml complete RPMI medium supplemented with 10 U/well murine recombinant IL-2 (R & D Systems, Abingdon, UK) and 3 µM Monensin (Sigma) either with or without peptide. The peptides were used at a concentration of 0.1 µg/ml (LCMV GP33–41) or 1 µg/ml (VSV NP52–59). After 5.5 h of culture, 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/Monensin 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 and resuspended in PBS with 0.05% saponin. After 10 min of incubation in the dark at 20°C, cells were pelleted and resuspended in PBS with 0.05% saponin and relevant antibodies. After incubation for 20 min at 4°C, cells were washed twice in saponin and analyzed.

Fractionation of cells
Splenocytes were sorted using a Becton Dickinson FACStar Plus (2,27). Post-sort analyses were performed to determine sort purity; the purity of all populations was >90%.

Cytotoxicity assays
The activity of cytotoxic T cells (CTL) was assessed in 51Cr-release assays (22,28). Targets for evaluation of LCMV-specific cytotoxicity were EL-4 cells pulsed with LCMV GP33–41 for 2 h; unpulsed EL-4 cells served as control targets. A redirected killing assay employing FcR-bound mAb to CD3{varepsilon} was used to detect total cytotoxic activity (29). Target cells were FcR+ P815 cells and P815 cells incubated with effector cells in the absence of mAb were used as negative control. Assay time was 5–6 h and the percentage of specific lysis was calculated as previously described (22).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Proliferation and phenotypic changes following LCMV infection or poly(I:C) injection.
Poly(I:C) has recently been used to model virus-induced T cell activation (1). For this reason we wanted to directly compare the activation status of CD8+ T cells during the course of a viral infection with that observed after injection of poly(I:C). Cell cycle progression as well as expression of activation markers were evaluated following these two different stimuli. Groups of mice were injected with either LCMV or poly(I:C), given BrdU in their drinking water for a period of 3 days prior to analysis, and on the indicated days, spleen cells were harvested and the CD8+ T cell subset phenotypically characterized. As shown in Fig. 1Go(A), CD8+ T cells proliferated after poly(I:C) injection to an extent comparable to that observed in LCMV-infected mice given BrdU on days 1–4 post-infection. Proliferation in both LCMV- and poly(I:C)-injected mice was restricted to cells with a relatively high expression of CD44. The massive proliferation seen on days 6 and 8 after virus infection was never observed in poly(I:C)-treated mice, not even when these were given poly(I:C) several times (data not shown). Figure 1Go(C) shows that splenic CD8+ cells in poly(I:C) injected mice retain a naive phenotype (L-selectinhiVLA-4lo), whereas in LCMV-infected mice most CD8+ cells gradually acquire an activated (L-selectinloVLA-4hi) phenotype. This difference in phenotype between poly(I:C)- and LCMV-injected mice was not apparent before day 6 in LCMV-infected mice, at which time point a marked down-regulation of L-selectin can be observed (Fig. 1BGo). Notably, injection of poly(I:C) does not lead to an up-regulation of IL-2R (1), whereas transient up-regulation is noted in LCMV-infected mice around this time point (2,27). Therefore, this could suggest that activation through this receptor might be important in deciding whether the initial activation should abort or continue. In support of this, a more detailed analysis revealed a clear-cut correlation between the appearance of IL-2R and phenotypic conversion (down-regulation of L-selectin) in LCMV-infected mice (Fig. 2Go).



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Fig. 1. Flow cytometric analyses of splenocytes from C57BL/6 mice infected with 103 LD50 of LCMV i.v. or injected with 150 µg of poly(I:C) i.p. Mice were given BrdU in their drinking water for 3 days and on the indicated days splenocytes were surface stained with: (A) PE-conjugated anti-CD44 and CyChrome-conjugated anti-CD8 or (B) PE-conjugated anti-CD8 and biotinylated anti-L-selectin followed by streptavidin–TriColor, then permeabilized and stained with FITC-conjugated anti-BrdU; gates were set for CD8+ cells. Figures in parentheses refer to the subset distribution in uninfected mice. In (C), cells were surface stained with PE-conjugated anti-CD8, FITC-conjugated anti-{alpha}4 integrin and biotinylated anti-L-selectin; gates were set for CD8+ cells.

 


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Fig. 2. IL-2R{alpha} (CD25) expression in C57BL/6 mice following infection with 103 LD50 of LCMV i.v. or injection of 150 µg poly(I:C) i.p. At the indicated days splenocytes were surface stained with PE-conjugated anti-CD8, FITC-conjugated anti-L-selectin and biotinylated anti-CD25 followed by streptavidin–TriColor; gates were set for CD8+ cells.

 
A marked difference was also observed with regard to functional activation (Fig. 3Go). Thus, in a redirected killing assay revealing all CTL irrespective of specificity, high levels of cytotoxicity were observed with splenocytes from virus-infected animals, whereas marginal cytotoxicity was noted following injection of poly(I:C)—even in old mice with increased numbers of CD44hi cells. Thus it appears that non-specific signals induced by agents like poly(I:C) are able to induce CD8+ T cells to enter cell cycle, but not to undergo functional differentiation. Notably, extending previous findings by Tough et al. (1), we could confirm that IFN-{alpha}/ß is a pivotal mediator involved in this partial T cell activation: no poly(I:C)-induced T cell proliferation was observed in IFN-{alpha}/ßR–/– mice (data not shown).



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Fig. 3. No functional activation of CD8+ T cells upon poly(I:C) injection. C57BL/6 were either injected i.p. with 150 µg poly(I:C) or given 150 µl of PBS as sham treatment. As a positive control, C57BL/6 were infected with 106 p.f.u. of VSV i.v. On day 3 [poly(I:C)] and day 6 (VSV) total CTL activity of spleen cells were tested in a redirected killing assay using anti-CD3 and FcR+ P815 as target cells. No lysis was observed in the absence of anti-CD3.

 
Poly(I:C) induces proliferation of already primed cells, but not of naive cells
Based on the finding that poly(I:C) shares some qualities with live virus and could initiate proliferation of CD8+CD44hi T cells, we wanted to characterize in greater detail this non-specific proliferative response. For this analysis, we wanted a system where the activation history of a sizeable subset of cells could be controlled and the cells could be identified in the flow cytometer. This was obtained through the use of cells from a mouse strain in which 50–60% of the CD8+ cells express a TCR directed against LCMV GP33–41 (19). These cells can be identified by mAb against V{alpha}2 and Vß8, and, moreover, the frequency of cells with endogenous V{alpha}2 is sufficiently low so that this antibody alone can be used for identification of TCR transgenic cells (26), allowing combinatorial analysis of the surface phenotype of these cells. To obtain uniformly primed LCMV TCR transgenic cells, wild-type C57BL/6 mice were transplanted with low numbers of transgenic cells and primed with LCMV ~30–40 days prior to injection of poly(I:C) (26). For the experiments, LCMV-primed and transplanted C57BL/6 mice, naive transgenic mice, and matched wild-type mice were injected with poly(I:C) and given BrdU for 3 days; BrdU incorporation and the expression of CD44 were evaluated (Fig. 4Go). As expected no difference in either CD44 expression or proliferation between CD8+V{alpha}2+ and CD8+V{alpha}2 T cells was observed in wild-type mice consistent with random low-grade priming due to stimulation from environmental antigens. A significant difference in the frequency of proliferating T cells in the CD8+V{alpha}2+ and CD8+V{alpha}2 subsets was observed in naive transgenic mice given poly(I:C); only CD8+V{alpha}2 cells proliferated to a significant degree and this difference in the level of proliferation correlated with a marked difference in CD44 expression as only CD8+V{alpha}2 T cells contained a CD44hi subset. In contrast to this, CD8+V{alpha}2+ cells present in transplanted and LCMV-primed C57BL/6 mice (being mostly LCMV TCR transgenic CD8+ T cells—as indicated by the finding that ~85% of these cells also express Vß8) proliferated extensively and in this case all CD8+V{alpha}2+ cells had a CD44int or CD44hi phenotype. These findings confirm a clear correlation between priming history, CD44 expression and proliferation in response to a non-specific stimulus, and raise the question whether similar non-specific proliferation of memory cells is induced in the context of a viral infection.



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Fig. 4. Correlation between priming status, CD44 expression and proliferation upon poly(I:C) injection. Transplanted and LCMV-primed C57BL/6, naive transgenic and wild-type mice were either left untreated (A) or injected with 150 µg of poly(I:C) i.p. and given BrdU in their drinking water for 3 days (B). Cells were surface stained with: (A) CyChrome-conjugated anti-CD8, PE-conjugated anti-CD44 and FITC-conjugated anti-V{alpha}2; (B) CyChrome-conjugated anti-CD8, PE-conjugated anti-V{alpha}2, permeabilized and stained with FITC-conjugated anti-BrdU. Gates were set for CD8+V{alpha}2+ and CD8+V{alpha}2cells, and the percentage of BrdU+ cells is presented; numbers in parentheses refer to BrdU+ cells in PBS-injected mice.

 
Limited proliferation of naive LCMV TCR transgenic cells upon infection with VSV
To answer the above question, we examined the response of LCMV TCR transgenic T cells in the context of infection with an unrelated virus, VSV. Like poly(I:C), VSV induces a transient IFN-{alpha} response (30) as evidenced here by the rapid up-regulation of Ly-6C on CD8+ T cells (not shown). Both the very early phase of infection (day 0–3) and the later phase (day 3–6) leading to the appearance of virus-specific CTL effectors were investigated. First, we evaluated the proliferative response of naive cells. Transgenic mice and matched wild-type mice were infected with VSV on day 0 and administered BrdU in their drinking water for a period of 3 days starting either at day 0 or day 3 post-infection On days 3 and 6 post-infection, spleen cells were harvested and analyzed by FACS (Fig. 5Go). As expected, no difference in the percentage of proliferating cells was observed between the CD8+V{alpha}2+ and CD8+V{alpha}2 subset in wild-type mice, neither in the early phase nor in the late phase. A significant difference was observed between the two subsets in transgenic mice. CD8+V{alpha}2 cells showed the same pattern of proliferation as CD8+ cells in wild-type mice, whereas the CD8+V{alpha}2+ subset proliferated to a much lesser extent, both in the very early and the later phase of infection. Notably, CD8+V{alpha}2+ cells responded vigorously to stimulation with the nominal antigen (data not shown).



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Fig. 5. Limited proliferation of naive transgenic cells. Transgenic mice and wild-type mice were infected with 106 p.f.u. of VSV i.v. and given BrdU for 3 days prior to analysis. On days 3 and 6 post-infection splenocytes were surface stained with CyChrome-conjugated anti-CD8 and PE-conjugated anti-V{alpha}2, permeabilized, and stained with FITC-conjugated anti-BrdU. Gates were set for CD8+V{alpha}2+ and CD8+V{alpha}2 cells, and the medians and ranges of BrdU+ cells are presented (n = number of animals tested).

 
Preferential phenotypic and functional activation of non-transgenic cells following VSV infection
We also evaluated the phenotypic changes induced by infection of naive transgenic mice with VSV 6 days earlier. No significant phenotypic differences were observed between the CD8+V{alpha}2+ and CD8+V{alpha}2 subsets in transgenic mice prior to VSV infection. After infection with VSV, no phenotypic changes were observed for the CD8+V{alpha}2+ subset as these cells retained a naive phenotype, VLA-4lo L-selectinhi. In contrast, many CD8+V{alpha}2 cells became phenotypically activated, up-regulating VLA-4 and down-regulating L-selectin (Fig. 6AGo).



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Fig. 6. Minimal phenotypic and functional activation of transgenic cells. Transgenic mice and wild-type mice were injected with 106 p.f.u. of VSV i.v. or given PBS as sham treatment. On day 6 post-infection splenocytes were surface stained with: (A) CyChrome-conjugated anti-CD8, PE-conjugated anti-V{alpha}2 and FITC-conjugated anti-{alpha}4-integrin; (B) FITC-conjugated anti-CD8, PE-conjugated anti-V{alpha}2 and biotinylated anti-L-selectin followed by streptavidin–TriColor. Gates were set for CD8+V{alpha}2+ and CD8+V{alpha}2 cells, and the expression of VLA-4 and L-selectin is depicted. In (C), splenocytes from C57BL/6 mice infected with 103 LD50 of LCMV i.v., naive transgenic mice and transgenic mice infected with 106 p.f.u. of VSV i.v. were tested for LCMV-specific cytotoxicity. Peptide-pulsed (GP33–41) EL-4 cells served as targets; unpulsed EL-4 cells were used as controls. In (D), total CTL activity of sorted CD8+V{alpha}2+ and CD8+V{alpha}2 spleen cells from day 6 VSV-infected transgenic mice was evaluated in a redirected killing assay using anti-CD3 and FcR+ P815 as target cells; no lysis was observed in the absence of anti-CD3.

 
Finally, we wanted to determine if LCMV TCR transgenic cells became functionally activated after infection with VSV. Transgenic mice were infected with VSV or given PBS and as a positive control we used C57BL/6 mice infected with LCMV. On day 6 (transgenic mice) or day 8 (LCMV-infected C57BL/6) spleen cells were tested in a 51Cr-release assay with GP33–41-pulsed EL-4 cells as targets. Limited functional activation of transgenic cells was observed (Fig. 6CGo). Although a peptide-specific lysis of 20–30% at the highest E:T ratio may appear to be significant, this should be seen in the light of the very high cytotoxicity observed with spleen cells from LCMV-infected wild-type mice. Given that the frequency of GP33–41-specific CD8+ T cells in transgenic mice is ~1/20 (19,26) and the frequency of GP33–41-specific effector cells in acutely infected wild-type mice has recently been determined to be only ~2-fold higher (i.e. ~1/10) (17,18), it can be calculated from the dose–response curves in the CTL assay that at most a few percent of naive LCMV TCR transgenic cells have acquired cytolytic capacity due to infection with an unrelated virus; this may reflect that some of these cells express an additional V{alpha} chain (14) and therefore potentially possess dual specificity. In keeping with these calculations, direct visualization of primed CD8+ T cells through detection of IFN-{gamma} intracellularly did not reveal any significant increase in GP33–41-specific IFN-{gamma}+ T cells following infection with VSV whereas about one-fifth of non-transgenic CD8+ cells reacted with the immunodominant VSV peptide (Fig. 7Go). To enable direct comparison of CTL activity on a per cell basis, spleen cells from day 6 VSV-infected transgenic mice were sorted into CD8+V{alpha}2+ and CD8+V{alpha}2 cells, and tested together with unsorted spleen cells in a redirected killing assay. From Fig. 6Go(D) it is obvious that the cytotoxicity observed in VSV-infected transgenic mice is almost exclusively limited to the non-transgenic cells. Overall, the above experiments clearly demonstrate that virus-induced bystander activation of naive non-cross-reacting T cells is very inefficient.



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Fig. 7. Intracellular IFN-{gamma} production in transgenic mice and wild-type mice infected i.v. with 106 of VSV or 103 LD50 of LCMV. On day 8 (LCMV) or 6 (VSV) post-infection, splenocytes were stimulated in vitro with either LCMV or VSV peptide for 5.5 h. Cells were surface stained with CyChrome-conjugated anti-CD8 and FITC-conjugated anti-V{alpha}2, permeabilized, and stained with PE-conjugated anti-IFN-{gamma}; gates were set for CD8+ cells. No IFN-{gamma}+ cells were detected without peptide stimulation.

 
Extensive proliferation of primed LCMV TCR transgenic cells early, but not late after infection with VSV
Since memory T cells differ from naive cells in their activation requirements (31), the activation of primed LCMV TCR transgenic T cells was also analyzed. Transplanted and LCMV-primed C57BL/6 mice were infected with VSV on day 0 and administered BrdU in their drinking water for a 3 day period starting either day 0 or 3 post-infection. Uninfected, but transplanted and LCMV-primed C57BL/6 were used as controls. Following previous priming with LCMV, both CD8+V{alpha}2+ and CD8+V{alpha}2 cells proliferated in the early phase of VSV infection (day 0–3 post-infection) and, similar to the pattern following injection of poly(I:C), a higher percentage of transgenic cells had incorporated BrdU, probably reflecting a more uniform priming status (see Figs 4 and 8GoGo). However, in the later phase of infection (day 3–6 post-infection) only limited proliferation of CD8+V{alpha}2+ T cells could be found, whereas CD8+V{alpha}2 T cells proliferated vigorously.



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Fig. 8. Proliferation of primed transgenic cells in the early phase of infection. Transplanted and LCMV-primed C57BL/6 mice were either infected with 106 p.f.u. of VSV i.v. or injected with 100 µg of GP33–41 i.p. Mice were given BrdU for a period of 3 days and splenocytes were analyzed by FACS. Cells were surface stained with CyChrome-conjugated anti-CD8 and PE-conjugated anti-V{alpha}2, permeabilized, and stained with FITC-conjugated anti-BrdU. Gates were set for CD8+V{alpha}2+ and CD8+V{alpha}2 cells, and medians and ranges of BrdU+ cells are presented (n = number of animals tested).

 
To exclude that the transient proliferation of primed transgenic CD8+ T cells could reflect that these cells were fully responsive to non-specific signals delivered early in the infection, but were anergic to stimulation through the TCR, transplanted and LCMV-primed C57BL/6 mice and naive wild-type mice were inoculated i.p. with 100 µg of LCMV GP33–41. Peptide was chosen for re-stimulation, because we knew from previous experience that a very high dose of LCMV would be required to induce substantial reinfection (32) and under such conditions also a strong non-specific stimulus would be delivered, making it impossible to discriminate between specific and non-specific activation. BrdU incorporation over a 3 day period was evaluated (Fig. 8Go). As expected LCMV TCR transgenic cells were clearly responsive to stimulation through the TCR and did not show any indication of being anergic. Notably, CD8+V{alpha}2 T cells proliferated only to a very limited extent upon peptide stimulation. Because more extensive proliferation was seen in LCMV-primed C57BL/6 mice that had not received LCMV TCR transgenic cells (not shown), this pattern is consistent with the prediction that in transplanted mice the transgenic CD8+V{alpha}2+ T cells dominate the GP33–41 response, and impede priming and/or proliferation of other GP33–41 specific clones (18).

Primed LCMV TCR transgenic cells do not become phenotypically nor functionally activated following infection with VSV
To see if VSV infection would induce phenotypic changes of primed LCMV TCR transgenic T cells, transplanted and LCMV-primed C57BL/6 mice were either infected with VSV or left untreated, and on day 3 (data not shown) and day 6 post-infection spleen cells were analyzed by flow cytometry. Little or no phenotypic change was observed for either CD8+ subpopulation on day 3 post-infection (not shown). However, on day 6 post-infection a marked down-regulation of L-selectin expression was observed for the CD8+V{alpha}2 subset, whereas no change in the expression of this molecule was observed for transgenic CD8+V{alpha}2+ T cells (Fig. 9Go). A shift from VLA-4lo to VLA-4hi was also observed for the CD8+V{alpha}2 subset on day 6 following VSV infection. A minor percentage of the CD8+V{alpha}2+ T cells also up-regulated VLA-4 upon infection with VSV, but the majority retained expression at the memory level which is in between that of naive cells and recently activated T cells. The fraction of CD8+V{alpha}2+ T cells that did change to a VLA-4hi phenotype nearly corresponded in size to a small subset of CD8+V{alpha}2+ cells that had retained a VLA-4lo phenotype in LCMV-primed mice. Therefore this phenotypic shift is likely to reflect de novo activation of recipient V{alpha}2+ T cells that have no specificity towards LCMV. Functionally, ~2-fold increase in CTL activity against GP33–41-coated targets was observed on day 3 post-infection following VSV challenge of transplanted and LCMV-primed mice (data not shown). Overall, these findings indicate that although LCMV-specific memory cells are induced to proliferate following challenge with an unrelated virus, little if any differentiation occurs in the absence of cross-reactivity at the TCR level.



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Fig. 9. Minimal phenotypic activation of primed transgenic cells. Transplanted and LCMV-primed C57BL/6 mice were either infected i.v. with 106 p.f.u. of VSV or left untreated as controls. On day 6 post-infection splenocytes were surface stained with: (A) CyChrome-conjugated anti-CD8, PE-conjugated anti-V{alpha}2 and FITC-conjugated anti-{alpha}4 integrin; (B) FITC-conjugated anti-CD8, PE-conjugated anti-V{alpha}2 and biotinylated anti-L-selectin followed by streptavidin–TriColor. Gates were set for CD8+V{alpha}2+ and CD8+V{alpha}2 cells, and the expression of VLA-4 and L-selectin is depicted.

 
Most CD8+ T cells responding in the early phase of VSV infection are stable during the subsequent phase of maximal virus-induced proliferation
To study the specificity and fate of CD8+ T cells in wild-type mice which proliferate early in the VSV infection, we applied a pulse–chase approach (1). Mice were infected with VSV and pulsed with BrdU in their drinking water for 3 days following which they were switched to normal water for 6 days; mice treated with poly(I:C) were included for comparison. Since VSV-induced proliferation peaks around day 5–6 post-infection, whereas poly(I:C) induces T cells to proliferate only for a brief period (<3 days), one would expect a marked difference in the half-life of BrdU-labeled CD8+ cells in the two situations, provided that most of the cells proliferating early in the infection were VSV specific. However, this was clearly not the case (Fig. 10Go); in fact most of the cells labeled during the pulse period survived the infection without undergoing substantial further proliferation or apoptosis (33,34).



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Fig. 10. C57BL/6 mice were infected with 106 p.f.u. of VSV i.v. or injected with 150 µg poly(I:C) i.p. and given BrdU in their drinking water day 0–3 post-infection. Mice were either analyzed on day 3 or placed on normal water and tested on day 9. Splenocytes were surface stained with CyChrome-conjugated anti-CD8 and PE-conjugated anti-CD44, permeabilized, and stained with FITC-conjugated anti-BrdU. Gates were set for CD8+ cells.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we have analyzed the capacity of a viral infection to induce non-specific activation of CD8+ T cells, particularly the ability to activate memory T cells for other viral infections. Two apparently opposing models have been used to explain the extensive CD8+ T cell activation often observed in the context of viral infections. One model predicts that there is little or no non-specific activation and if only all viral epitopes are included in the analysis, all activated T cells can be accounted for. This view has recently received strong support from studies analyzing the T cell response using tetramer staining (17). On the other hand, the IFN-inducer poly(I:C) causes non-specific activation of memory T cells, suggesting that viruses which induce a significant production of this cytokine would have the same effect (1). The findings in the present report make it possible to reconcile these different views indicating that both mechanisms are at work during viral infection and that the outcome of any analysis is very dependent on the timing of evaluation as well as the parameter evaluated. Thus our results provide evidence that the marked proliferation of CD8+ T cells observed in many viral infections is composed of at least two components. In the early phase of infection, non-specific stimuli [e.g. IFN-{alpha}/ß, but other cytokines may have similar effect (12)] lead to a preferential proliferation of already primed (CD44hi) CD8+ T cells. In the later phase of infection, TCR ligation is necessary to induce proliferation and further differentiation.

Transgenic mice in which ~50% of the CD8+ T cells express a TCR specific for GP33–41 of LCMV were selected as our model system based on the following considerations: This transgenic model provides us with a well-defined population of cells with known specificity which easily can be detected by the use of mAb directed towards the transgenic TCR. As only 50% of the CD8+ T cells express the transgenic TCR, activation of these cells can be investigated in a reasonably balanced milieu and the non-transgenic cells (CD8+V{alpha}2) provide an internal control. Similarly, memory cells with the same specificity and constituting about the same fraction of the CD8+ population can be generated by transfer of LCMV TCR transgenic cells into wild-type mice followed by LCMV infection.

The early phase of a viral infection can be mimicked by injection of poly(I:C), which is a potent inducer of IFN-{alpha}/ß (35,36). Our initial experiments with poly(I:C) confirmed recent findings by Tough et al. demonstrating that poly(I:C) induces transient proliferation of primed CD8+ T cells (CD44hi) (1). However, this non-specific signal does not induce phenotypic or functional activation. As many viral infections are associated with substantial production of IFN-{alpha}/ß, this led us to presume that the early phase of virus-induced CD8+ T cell proliferation regarding primed cells (CD44hi) would resemble poly(I:C)-induced proliferation. Indeed, extensive proliferation of primed LCMV-specific transgenic cells was observed in the early phase of a VSV infection. Notably, this infection is associated with an IFN-{alpha}/ß response very similar to that induced by poly(I:C) (30), but unfortunately the involvement of this cytokine in the response of infected animals cannot be directly proven as VSV-infected IFN-{alpha}/ßR–/– mice succumb before day 3 post-infection (unpublished observation). Despite the massive proliferation, little or no phenotypic and functional activation was induced. In contrast to primed cells, naive transgenic cells did not proliferate in the early phase of infection, which is consistent with the notion that primed T cells are more easily activated and may express cell surface receptors absent on naive cells (31).

Regarding the late phase of infection, our results clearly indicate that some degree of antigen-specific triggering through the TCR is necessary to induce both proliferation and further differentiation. Even in the context of a vigorous immune response towards VSV during which the majority of non-transgenic cells became activated, little if any activation of transgenic cells was observed, even in the case of memory cells. This lack of activation was not due to transgenic cells being anergic to triggering through their TCR as they proliferated extensively upon stimulation with their nominal peptide. Consequently, it may be concluded that virus-induced activation of naive cells as well as functional activation of heterologous memory T cells requires TCR–ligand interaction. From this follows that induction of cytolytic activity in memory cells primed through previous exposure to unrelated agents is restricted to cells which are at least partially cross-reactive at the TCR level.

Two recent reports also employing TCR transgenic mice to study virus-induced bystander activation contain observations which basically are in keeping with the conclusions reached here, although neither reveals the complete picture. Thus, using mice in which most CD8+ T cells are LCMV specific, Ehl et al. found limited functional activation of naive transgenic cells following infection with vaccinia virus (14). However, this is not an ideal model and only the responsiveness of naive cells was investigated, and therefore it could not be excluded that memory cells might be activated. Moreover, only effector cell capacity (cytolytic activity) was evaluated, whereas we have included cell cycle progression as parameter of activation. Recently, Zarozinski et al. also studied bystander activation and concluded that neither proliferation nor differentiation of irrelevant T cells was induced (37). In that study the activation of both naive and primed cells was analyzed. However, only the response at the time of normal peak activity was assessed and this probably explains why they did not detect any non-specific proliferation which may be missed unless earlier time points are also analyzed. Therefore, our study underscores the importance of evaluating several parameters of T cell activation (proliferation, phenotypic conversion, cytolytic activity) (21) and more than one time point.

From a functional point of view, the evolutionary advantage of a discrimination between stimuli required to induce proliferation of primed cells and stimuli necessary for further differentiation is obvious. Thus, in the context of a viral infection, memory to heterologous antigens can be sustained, and at the same time unwanted and potentially dangerous induction of irrelevant cytotoxic effector T cells is avoided. If memory cells were simply long-lived, non-dividing cells they would rapidly be diluted out during subsequent infections with unrelated agents. Our findings together with those of Tough et al. (1) suggest a mechanism which allows the persistence of memory T cells independent of the specific antigenic environment, but in balance with the overall microbial load. Consequently cytokine-induced proliferation could be very important for long-term maintenance of an expanded pool of memory CTLp cells. The extent of non-specific proliferation of memory cells will depend on the amounts and nature of the cytokines induced during the viral infection. Although IFN-{alpha}/ß play a role in inducing proliferation of memory T cells, it may not be the only cytokine with this function (12). Indeed, recent results indicate that IL-15 may be more central to this response (38).

The fact that antigen-specific TCR triggering is required to induce CD8+ cells to become cytotoxic effectors limits the risk of unwanted activation of, for instance, autoimmune effector cells (39). Only memory cells which are cross-reactive with the infecting agent will be driven to become fully differentiated effector cells.

In conclusion, our findings indicate that both non-specific and virus-specific stimuli contribute to virus-induced proliferation of CD8+ T cells. Which component dominates depends on the time point of investigation; in the early phase of infection primed CD8+ T cells proliferate in response to non-specific stimuli (e.g. IFN-{alpha}/ß), whereas the later phase is virus specific, and TCR-ligation is required for proliferation and differentiation. That viruses induce non-specific proliferation of memory T cells may play a role in maintaining an expanded memory pool even in the absence of cross-reactivity.


    Acknowledgments
 
The authors wish to thank S. Buus for providing LCMV and VSV peptides, and H. Pircher and R. M. Zinkernagel for providing us with breeder pairs of the LCMV TCR transgenic line used in this study. This work was supported in part by the Danish Medical Research Council, the Biotechnology Center for Cellular Communication, and the Novo Nordisk Foundation. J. P. C. was the recipient of a PhD scholarship from the Faculty of Health Sciences, University of Copenhagen.


    Abbreviations
 
CTLcytotoxic T lymphocyte
GP33–41amino acids 33–41 of LCMV glycoprotein I
IFN-{alpha}/ßR–/–type I IFN receptor knockout
LCMVlymphocytic choriomeningitis virus
PEphycoerythrin
poly(I:C)polyinosinic–polycytidylic acid
VSVvesicular stomatitis virus

    Notes
 
1 Present address: Department of Immunology, St Jude Children's Research Hospital, North Lauderdale, Memphis, TN 38105, USA Back

Transmitting editor: A. McMichael

Received 15 March 1999, accepted 21 May 1999.


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