TCR down-regulation boosts T-cell-mediated cytotoxicity and protection against poxvirus infections

TCR down-regulation boosts T-cell-mediatedcytotoxicity and protection against poxvirus infections

Ann K. Hansen1, Matthias Regner2, Charlotte M. Bonefeld1,

Lasse Boding1, Martin Kongsbak1, Niels Ødum1,3, Arno Mullbacher2,

Carsten Geisler1 and Marina R. von Essen1

1 Department of International Health, Immunology and Microbiology, Faculty of Health

Sciences, University of Copenhagen, Copenhagen, Denmark2 Viral Immunology Group, Department of Emerging Pathogens and Vaccines, The John Curtin

School of Medical Research, Australian National University, Canberra, Australia3 Department of Biology, Faculty of Science, University of Copenhagen, Copenhagen, Denmark

Cytotoxic T (Tc) cells play a key role in the defense against virus infections.

Tc cells recognize infected cells via the T-cell receptor (TCR) and subsequently

kill the target cells by one or more cytotoxic mechanisms. Induction of the cytotoxic

mechanisms is finely tuned by the activation signals from the TCR. To determine

whether TCR down-regulation affects the cytotoxicity of Tc cells, we studied TCR down-

regulation-deficient CD3cLLAA mice. We found that Tc cells from CD3cLLAA mice have

reduced cytotoxicity due to a specific deficiency in exocytosis of lytic granules.

To determine whether this defect was reflected in an increased susceptibility to virus

infections, we studied the course of ectromelia virus (ECTV) infection. We found that the

susceptibility to ECTV infection was significantly increased in CD3cLLAA mice with a

mortality rate almost as high as in granzyme B knock-out mice. Finally, we found

that TCR signaling in CD3cLLAA Tc cells caused highly increased tyrosine phosphorylation

and activation of the c-Cbl ubiquitin ligase, and that the impaired exocytosis of lytic

granules could be rescued by the knockdown of c-Cbl. Thus, our work demonstrates that

TCR down-regulation critically increases Tc cell cytotoxicity and protection against

poxvirus infection.

Key words: c-Cbl . cytotoxicity . T cells . TCR down-regulation . virus

Supporting Information available online

Introduction

CD81 T cells are key components of the immune system and

undergo massive expansion in numbers during the acute phase of

infection [1]. This expansion serves to generate a large number of

cytotoxic T (Tc) cells that contribute to the recovery of the host

from the infection. Tc cells recognize specific antigen/MHC-I

complexes at the surface of infected cells by the use of the T-cell

receptor (TCR). Following recognition, the Tc cells can kill

infected target cells via three different pathways, namely (a) the

perforin/granzyme, (b) the Fas/Fas ligand (FasL), and/or (c) the

cytokine pathways [2].

Perforin and granzymes are located in specialized secretory

lysosomes in the Tc cell [3, 4]. Shortly after recognition of theCorrespondence: Prof. Carsten Geislere-mail: [email protected]

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

DOI 10.1002/eji.201141413 Eur. J. Immunol. 2011. 41: 1948–1957Ann K. Hansen et al.1948

target cell, the Tc cell polarizes its secretory machinery toward

the point of contact with the target cell [5]. The centrosome

relocates to the immunological synapse formed between the Tc

cell and the target cell, and the secretory lysosomes, frequently

called cytotoxic or lytic granules, move along the microtubules

toward the centrosome [6]. Here they dock at the plasma

membrane and deliver their content by exocytosis into

the small secretory cleft of the immunological synapse [7, 8].

Perforin and granzymes subsequently act in concert to induce

apoptosis of the target cell [9]. The Fas/FasL pathway requires

the interaction of FasL (CD178 or CD95L) on the Tc cell with Fas

(CD95) on the target cell. Fas signaling is mediated via death

domains leading to the induction of the caspase cascade, which

ultimately results in apoptosis of the target cell [2]. Because Fas is

ubiquitously expressed, the expression of FasL on Tc cells must be

tightly regulated. Although debated, the intracellular stores of

FasL in Tc cells are probably distinct from the lytic granules

containing perforin and granzymes [10, 11]. The perforin/gran-

zyme and the Fas/FasL pathways account for most of the

destruction of target cells by Tc cells. However, Tc cells also

produce several cytokines, such as IFN-g and TNF-a, which

have cytotoxic action when secreted in the vicinity of target cells

[12, 13].

The cytotoxic pathways used by Tc cells are all dependent on,

and finely tuned by, TCR signaling [14–16]. As many other cell

surface receptors, the TCR is down-regulated following ligand

triggering. At least two distinct pathways exist for TCR down-

regulation [17, 18]. One pathway is dependent on protein tyro-

sine kinase and Cbl activity and leads to TCR ubiquitination and

degradation [19, 20]. The other pathway is dependent on PKC-

mediated activation of the di-leucine-based (diL) motif found in

the CD3g chain of the TCR and leads to TCR recycling. The CD3gdiL motif plays a unique role in TCR trafficking, and the motif has

been characterized in detail at the molecular and cellular level

[21–28]. It is still not known why T cells need two distinct

pathways for TCR down-regulation, but they probably act in

concert to fine-tune TCR signaling and thereby T-cell effector

functions. To study the physiological roles of TCR down-regula-

tion mediated by the CD3g diL motif, we have generated

CD3gLLAA knock-in mice homozygous for a double leucine to

alanine mutation in the CD3g diL motif [29].

The aim of the present study was to investigate whether

TCR down-regulation mediated by the CD3g diL motif affects Tc

cell effector functions and thereby susceptibility to virus infec-

tions in a biological relevant viral infection model, namely

mousepox.

Results

Generation and characterization of Tc cells from P14and P14LLAA mice

We have previously described the generation of the CD3gLLAA

mice and shown that CD3gLLAA T cells have impaired PKC-

induced TCR down-regulation, reduced ligand-induced TCR

down-regulation and constitutive TCR cycling, and that their

TCR cell surface expression levels are increased by approximately

10% compared with T cells from wild-type mice [29]. To study Tc

cells with identical specificity, we crossed the CD3gLLAA mice to

the TCR transgenic P14 mice to generate P14LLAA mice. P14 and

P14LLAA TCR transgenic mice express a TCR (Va21Vb81)

specific for lymphocytic choriomeningitis virus (LCMV) glyco-

protein (gp33–41) bound to H-2Db [30, 31]. To generate antigen-

specific Tc cells, we stimulated splenocytes from P14 and

P14LLAA mice with LCMV glycoprotein gp33–41 for 3 days. The

cells were subsequently washed and then rested for 2 days

without peptide. Following this stimulation schedule, the

expanded CD81 cells constituted 95–98% of the total cell

numbers (Fig. 1A), and of these �99% were TCR transgenic

(Fig. 1B). CD81 T cells start to express perforin and granzymes

and up-regulate the expression of CD44 during their differentia-

tion into effector Tc cells. To determine the activation status of

the expanded CD81 Tc cells, we measured the expression levels

of intracellular granzyme B, perforin, and cell surface expressed

CD44. All of the expanded TCR transgenic Tc cells from P14 and

P14LLAA mice expressed granzyme B and perforin and high

levels of CD44 (Fig. 1C and D). Importantly, P14 and P14LLAA Tc

cells expressed equivalent levels of perforin and granzyme B. As

previously found for naıve CD3gLLAA T cells [29, 32], PKC-

induced TCR down-regulation was abolished in P14LLAA Tc cells

(Fig. 1E) and their TCR expression levels were increased

by approximately 10% compared with Tc cells from P14 mice

(Fig. 1F).

Reduced cytotoxicity and granzyme B exocytosis inP14LLAA Tc cells

The highly enriched populations of Tc effector cells obtained from

P14 and P14LLAA mice after the in vitro expansion described

above allowed us directly to compare their cytolytic potential. We

incubated equal numbers of the in vitro generated P14 and

P14LLAA Tc cells with EL-4 target cells that were either pulsed

with gp33–41 or mock-pulsed. We found that P14LLAA Tc cells had

weakened cytolytic potential compared with P14 Tc cells. Thus,

for all effector/target cell ratios tested the cytotoxicity was

reduced by 30–35% for P14LLAA compared with P14 Tc cells

(Fig. 2A). To distinguish between perforin/granzyme- and Fas/

FasL-mediated cytolysis, we next performed cytotoxic assays in

the presence of concanamycin A that inhibits exocytosis of lytic

granules but leaves Fas/FasL-mediated cytotoxicity unaffected

[33]. We found that concanamycin A treatment significantly

inhibited the cytotoxicity of both P14 and P14LLAA Tc cells

(Fig. 2B). The residual cytotoxicity after concanamycin A

treatment did not significantly differ between P14 and P14LLAA

Tc cells. These results indicated that perforin/granzyme-

mediated cytotoxicity was not as efficient in P14LLAA as in P14

Tc cells. This could either be caused by a reduced amount of

perforin/granzyme inside P14LLAA Tc cells, impaired exocytosis

Eur. J. Immunol. 2011. 41: 1948–1957 Immunity to infection 1949


of the lytic granules, or a combination of the two. By intracellular

staining, we found that P14LLAA and P14 Tc cells contained

equal levels of perforin and granzyme B (Fig. 1C and D). In

contrast, we found that exocytosis of granzyme B was reduced by

30–35% in P14LLAA compared with P14 Tc cells (Fig. 2C). By

analogy, this means that the presence of an intact CD3g diL motif

Figure 1. Generation and characterization of Tc cells from P14 and P14LLAA mice. Splenocytes from P14 and P14LLAA mice were stimulated for3 days with gp33–41, washed, rested for 2 days, and subsequently analyzed for (A) expression of CD81, (B) expression of the Va2Vb8 transgenic TCR,(C) expression of CD44 and intracellular granzyme B, and (D) expression of CD44 and intracellular perforin. The MFIs of the granzyme B andperforin staining are given in the upper right quadrant of the respective dot plots. (E) PKC-induced TCR down-regulation. P14 Tc cells (filledsquares) and P14LLAA Tc cells (open squares) were plated with 50 nM of phorbol 12,13-dibutyrate at 371C for the time indicated. The cells weresubsequently analyzed by flow cytometry and the degree of TCR down-regulation calculated. The data show mean values7SEM obtained fromthree independent experiments. (F) TCR expression levels. P14 and P14LLAA Tc cells were stained with mAb against CD8 and Va2. The Va2expression levels were subsequently determined on CD81Va21 T cells. The black curve represents P14 and the gray curve P14LLAA Tc cells. TheMFI of the Va2 staining and the relative TCR expression level on P14LLAA cells are given; ��po0.005. (A–F) Data are representative of at least threeindependent experiments. The full gating strategy used in this study is shown in Supporting Information Fig. 1.

Figure 2. Characterization of Tc cell cytotoxicity. (A and B) In vitro generated P14 (filled symbols) and P14LLAA (open symbols) Tc cells wereincubated with 51Cr-labeled gp33–41 pulsed (diamonds) or mock-pulsed (squares) EL-4 cells at the indicated effector/target cell ratios for 5 h andspecific 51Cr release was subsequently determined. In (B) the Tc cells were pre-incubated with 100 nM concanamycin A. (C) Granzyme B secretion.P14 (filled bars) and P14LLAA (open bars) Tc cells were plated with gp33–41 pulsed splenocytes for the time indicated and the amount of granzyme Bin the supernatant was subsequently determined by ELISA. (D–G) Production and release of IFN-g and TNF-a. P14 (filled bars) and P14LLAA (openbars) Tc cells were plated with gp33–41 pulsed splenocytes for (D and F) 4 h or (E and G) the time indicated. The percentages of (D) IFN-g1 and (F)TNF-a1 cells were determined by intracellular staining, and the amount of secreted (E) IFN-g and (G) TNF-a was determined by ELISA. (A–G) Thedata show mean values7SEM obtained from two to five independent experiments; �po0.05; ��po0.005. Statistical analyses were performed usingStudent’s t-test with a 5% significance level, unpaired observations, and equal variance.

Eur. J. Immunol. 2011. 41: 1948–1957Ann K. Hansen et al.1950


in P14 Tc cells increases exocytosis of lytic granules and

cytotoxicity by �50% compared with P14LLAA Tc cells. To

determine whether other cytolytic pathways were affected by the

CD3g diL motif, we next determined IFN-g and TNF-a production

and secretion following stimulation with gp33–41 pulsed spleno-

cytes. We found that P14 and P14LLAA Tc cells produced and

secreted similar amounts of IFN-g and TNF-a (Fig. 2D–G).

Finally, we measured the expression of FasL at the surface of the

Tc cells during co-culturing with gp33–41 pulsed target cells and

found that P14 and P14LLAA Tc cells expressed similar levels of

FasL (Supporting Information Fig. 2).

Impaired protection against poxvirus infections inCD3cLLAA mice

The data above indicated that perforin/granzyme-mediated Tc cell

cytotoxicity in vitro is enhanced by approximately 50% in Tc cells

with an intact CD3g diL motif compared with Tc cells that lack a

functional CD3g diL motif. To determine whether this boost in

cytotoxicity was reflected in an increased protection against acute

virus infections, we next studied the course of ectromelia virus

(ECTV) infection in C57BL/6, CD3gLLAA, and granzyme B knock-

out (gzmB–/–) mice. ECTV is a natural mouse pathogen that causes

mousepox, and resistance to ECTV is highly dependent on perforin/

granzyme-mediated cytotoxicity [34–37]. We infected mice with

1� 103 PFU of virulent Moscow ectromelia virus (Mo-ECTV) s.c.

and closely followed the morbidity and the mortality of the mice for

21 days. We found that the mortality rate of CD3gLLAA mice

following Mo-ECTV infection was significantly (po0.0001)

increased compared with C57BL/6 mice and almost as high as

the mortality rate of gzmB–/– mice (Fig. 3A). To further analyze the

disease progression, we determined the virus load in the spleens of

mice 4 and 6 days after infection. Four days after infection, the

virus titers in CD3gLLAA and C57BL/6 spleens were similar,

whereas they were significantly increased in gzmB–/– spleens

(Fig. 3B). At this early time point, the innate immune system and

especially the natural killer (NK) cells play a key role in clearance of

the virus [38]. Thus, the virus titers observed at day 4 were in

agreement with the reduced cytotoxic capacity of gzmB–/– NK cells

and furthermore indicated that the innate immune defense against

ECTV is intact in CD3gLLAA mice as would be expected. At day 6,

virus-specific Tc cells play an important role in the control of

poxvirus infections. At this time point, the virus titers in CD3gLLAA

and gzmB–/– spleens were significantly increased compared with

C57BL/6 spleens (Fig. 3B) in agreement with the increased

mortality rate observed in CD3gLLAA and gzmB–/– mice.

These observations strongly indicate that the CD3g diL motif

critically enhances Tc cell cytotoxicity in vivo. The deficient Tc

cell response in CD3gLLAA mice could be caused by a reduced

number of virus-specific Tc cells or by a defect in the cytolytic

mechanisms of the Tc cells. We consequently measured the total

number of CD81 cells and virus-specific B8R20–27-Kb-tetramer

positive (Ev-Kb-tet1) Tc cells in the spleens 6 days after infection

with ECTV. We found that the number of CD81 and Ev-Kb-tet1 Tc

cells was increased in CD3gLLAA mice compared with C57BL/6

mice (Fig. 3C and D). Thus, a reduced number of Tc cells did not

account for the increased susceptibility to ECTV in CD3gLLAA

mice. We subsequently determined the cytotoxic potential of

virus-specific Tc cells in spleens isolated 6 days after infection

with attenuated ECTV (Hampstead Egg ectromelia virus (HE-

ECTV)). We used HE-ECTV as this ECTV strain does not cause as

extensive tissue damage as Mo-ECTV. The capacity to kill ECTV-

infected mouse embryo fibroblasts (MEFs) was reduced by

approximately 30% in CD3gLLAA compared with C57BL/6 Tc

cells (Fig. 3E). Non-infected MEF targets were not killed by

C57BL/6 Tc cells (Fig. 3E black, filled squares), and pre-incuba-

tion of the Tc cells with concanamycin A completely inhibited

killing of ECTV-infected MEF (Fig. 3F). When HE-ECTV was used

for infection, the percentages and thereby the numbers of CD81

and Ev-Kb-tet1 Tc cells used in the in vitro cytotoxic assay were

similar for the three mouse strains analyzed (Fig. 3G and H, and

Supporting Information Fig. 3). As IFN-g, in addition to the

perforin/granzyme pathway, plays a role in survival following

ECTV infections, we also measured antigen-specific IFN-g secre-

tion. We found that splenocytes from CD3gLLAA and C57BL/6

mice produced similar amounts of IFN-g when stimulated with

ECTV-infected MEFs (Supporting Information Fig. 4).

Taken together, these experiments demonstrated that

CD3gLLAA mice are more susceptible to poxvirus infections than

C57BL/6 mice. This was not due to a reduced generation of

Tc cells but most probably to a deficiency in the perforin/gran-

zyme cytotoxic pathway of the CD3gLLAA Tc cells. This is in

agreement with the in vitro data demonstrating reduced cyto-

toxicity of P14LLAA Tc cells caused by a specific deficiency in

exocytosis of lytic granules.

Increased activation of c-Cbl inhibits exocytosis ofperforin/granzyme B in P14LLAA Tc cells

Several studies have demonstrated that both the strength and the

kinetics of TCR signaling affect Tc cell cytotoxicity [14–16].

Furthermore, it has recently been reported that the c-Cbl ubiquitin

ligase inhibits exocytosis of lytic granules and cytotoxicity in NK

cells [39]. To investigate whether the activity of c-Cbl was affected

in P14LLAA Tc cells, we determined the levels of tyrosine

phosphorylation of c-Cbl following stimulation of P14 and

P14LLAA Tc cells with gp33–41. We found significantly increased

c-Cbl tyrosine phosphorylation in P14LLAA Tc cells compared with

P14 Tc cells (Fig. 4A and B). Activated c-Cbl ubiquitinates the TCR,

which is subsequently internalized and degraded [18]. Thus, an

enhanced c-Cbl activity in P14LLAA Tc cells would be expected to

cause a greater degree of TCR degradation following TCR

triggering. In agreement, we found that degradation of the TCR

was significantly increased in P14LLAA compared with P14 Tc cells

(Fig. 4C and D). In NK cells, c-Cbl inhibits cytotoxicity by

inhibition of Vav1 signaling and this is probably mediated by

targeting of Vav1 for ubiquitylation [39]. In accordance with an

increased c-Cbl activity in P14LLAA Tc cells, we noticed increased

Vav1 degradation following TCR stimulation in P14LLAA Tc cells



as measured by reduced intensity of the Vav1 (95 kDa) bands

(Supporting Information Fig. 5). Taken together, these results

demonstrated an increased activity of c-Cbl in P14LLAA compared

with P14 Tc cells following TCR signaling. To directly determine

whether c-Cbl affected exocytosis of the lytic granules, we next

studied siRNA-mediated knockdown of c-Cbl (Fig. 4E). Whereas

control siRNA did not affect granzyme B exocytosis following TCR

triggering (Fig. 4F compared with Fig. 2C), c-Cbl knockdown

completely normalized granzyme B exocytosis in P14LLAA Tc cells

(Fig. 4F). Analyses of the same cell supernatants demonstrated the

c-Cbl knockdown did not affect secretion of IFN-g (Fig. 4F).

Discussion

In this study we report that TCR down-regulation mediated by the

CD3g diL motif plays a critical role for Tc cell cytotoxicity and

protection against poxvirus infections. We found that the reduced

cytotoxicity of Tc cells with a mutated CD3g diL motif was caused

by a reduced secretion of perforin/granzyme, whereas the IFN-g/

TNF-a pathway was unaffected in agreement with the existence of

separate secretory pathways for secretory lysosomes and cytokines

[40, 41]. Our study suggested that the Fas/FasL pathway is intact

in CD3gLLAA Tc cells, but further studies, like using Fas–/– target

cells, are required to draw a firm conclusion on this matter.

The presence of an intact CD3g diL motif in P14 Tc cells boosts

exocytosis of lytic granules and cytotoxicity by �50% compared

with P14LLAA Tc cells with a mutated CD3g diL motif. To

determine whether this CD3g diL motif-mediated enhancement

of the cytotoxic capacity played any physiological role we studied

the cause of poxvirus infections. We find that this is a suitable

model as resistance to the poxvirus ECTV is strictly dependent on

perforin/granzyme-mediated cytotoxicity [34–37]. We found

that CD3gLLAA mice were deficient in controlling ECTV infec-

tions and had significantly increased morbidity and mortality

compared with C57BL/6 mice. These observations substantiate

that the CD3g diL motif plays an important physiological role in

perforin/granzyme-mediated Tc cell cytotoxicity. At first sight it

Figure 3. Increased susceptibility to poxvirus in CD3gLLAA mice. (A) C57BL/6 (B6; n 5 27; black diamonds), CD3gLLAA (LLAA; n 5 35; whitediamonds), and granzyme B knock-out (gzmB–/–; n 5 37; white squares) mice were infected with 1�103 PFU Mo-ECTV s.c. in the hind leg. Mice weremonitored daily for 21 days; C57BL/6 versus CD3gLLAA, po0.0001; CD3gLLAA versus gzmB–/–, p 5 0.042. (B) Spleen virus titers given as log10 PFU/mLspleen lysate on days 4 and 6 from B6 (black bars), LLAA (white bars), and gzmB–/– (striped bars) mice infected by injection of 1�103 PFU Mo-ECTV.(C and D) Total number of (C) CD81 and (D) Ev-Kb-tet1 cells in spleens from B6 (black bars), LLAA (white bars), and gzmB–/– (striped bars) mice 6days after infection with 1�103 PFU Mo-ECTV. (E and F) Splenocytes isolated from B6 (black diamonds), LLAA (white diamonds), and gzmB–/–

(white squares) mice 6 days after i.v. infection with 1�104 PFU HE-ECTV were incubated with 51Cr-labeled MEF infected with Mo-ECTV or with non-infected MEF (black squares) at the indicated effector/target cell ratios for 5 h and specific 51Cr release was subsequently determined. In (F) theeffector cells were pre-incubated with 100 nM concanamycin A. Fraction of (G) CD81 and (H) Ev-Kb-tet1 cells in spleens from B6 (black bars), LLAA(white bars), and gzmB–/– (striped bars) mice 6 days after i.v. infection with 1�104 PFU HE-ECTV. (B–H) The data show mean values7SEM obtainedfrom one representative experiment of two to five experiments with three mice in each group; �po0.05. Statistical analyses were performed usingStudent’s t-test with a 5% significance level, unpaired observations, and equal variance. For the analysis of the survival data, we used the log-rank/Mantel–Cox test.



seemed surprising that the CD3gLLAA mice were almost as

susceptible to poxvirus infections as the gzmB–/– mice. However,

the exocytosis of lytic granula is intact in gzmB–/– Tc cells. Thus,

perforin and all other granzymes except for granzyme B are

normally secreted in gzmB–/– mice. In contrast, exocytosis of lytic

granula is impaired in CD3gLLAA Tc cells meaning that all of the

cytotoxic substances of the lytic granula are less efficiently

secreted in CD3gLLAA compared with gzmB–/– mice.

So why is perforin/granzyme-mediated Tc cell cytotoxicity

impaired in CD3gLLAA mice? We have previously shown that

constitutive TCR endocytosis is reduced in T cells from CD3gLLAA

mice [29], and in accordance, we found that TCR expression

levels on Tc cells from P14LLAA mice are increased by approxi-

mately 10% compared with P14 Tc cells. If no compensatory

mechanisms were found, a higher TCR expression level would be

expected to result in increased TCR signaling [42]. In agreement,

we have previously shown that early TCR signaling as measured

by tyrosine phosphorylation of ZAP-70, LAT, and PLC-g1 is

increased in P14LLAA T cells compared with P14 T cells [29]. At

first sight, this suggested that P14LLAA Tc cells should show

enhanced cytotoxicity [14–16]. However, concomitant with the

increased early TCR signaling we noticed a significantly increased

activation of c-Cbl in P14LLAA Tc cells. c-Cbl is known to be a

negative regulator of receptor signaling [20, 43, 44], and it has

recently been described that activation of c-Cbl inhibits exocytosis

of lytic granules and cytotoxicity in NK cells via inhibition of Vav1

[39]. Thus, it seemed likely that the increased activation of c-Cbl

in CD3gLLAA Tc cells impaired the exocytosis of lytic granules. In

Figure 4. Increased c-Cbl activation in CD3gLLAA Tc cells. (A) P14 and P14LLAA Tc cells were stimulated with gp33–41 for the time indicated. Thecells were lysed and analyzed for c-Cbl-specific tyrosine phosphorylation. Protein loading was quantified by probing the stripped membranes fortotal c-Cbl. (B) Semi-quantification of c-Cbl phosphorylation in P14 (black bars) and P14LLAA (white bars) Tc cells. The ordinate shows thenormalized, relative density of the c-Cbl-pY bands calculated as (c-Cbl-pY band densityt 5 x/P14 c-Cbl-pY band densityt 5 0). (A and B) Data arerepresentative of five independent experiments. (C) TCR degradation. P14 and P14LLAA Tc cells were biotinylated and stimulated with gp33–41 forthe indicated time. Biotinylated CD3e was determined by specific anti-CD3e antibodies (C, upper panel). Protein loading was quantified by probingthe stripped membranes with HRP-coupled streptavidin (C, lower panel). (D) TCR degradation was calculated for P14 (black bars) and P14LLAA(white bars) Tc cells as (CD3e band density/streptavidin band density)t 5 x and the values normalized setting t 5 0 to 100%. (E) c-Cbl knockdown.Total lysates of P14 and P14LLAA Tc cells 24 h after transfection with either control siRNA or siRNA specific for c-Cbl were immunoblotted for c-Cbland GAPDH. (F) Twenty-four hours after transfection with either control siRNA or siRNA specific for c-Cbl P14 (filled bars) and P14LLAA (open bars)Tc cells were plated with gp33–41 pulsed splenocytes for 4 h, and the amounts of granzyme B and IFN-g in the supernatants were subsequentlydetermined by ELISA. (A–C, E) Data are representative of three to five independent experiments. (D and F) The data show mean values7SEMobtained from three independent experiments; �po0.05; ��po0.005. Statistical analyses were performed using Student’s t-test with a 5% significancelevel, unpaired observations, and equal variance.



support of this assumption, knockdown of c-Cbl increased

exocytosis of lytic granules in both P14 and P14LLAA Tc cells and

normalized the exocytosis of lytic granules in P14LLAA Tc cells as

measured by secretion of granzyme B. These observations indi-

cate that the disturbed trafficking and expression of the TCR in

T cells without a CD3g diL motif cause an enhanced and not well-

coordinated TCR signaling resulting in an increased activation of

the negative regulator c-Cbl, and consequently we suggest that

CD3g diL motif-mediated TCR down-regulation increases Tc

cytotoxicity by weakening the activation of c-Cbl.

We do not have the full explanation why the expansion of

virus-specific Tc cells is unaffected or even increased in CD3gLLAA

mice following poxvirus infection but reduced following infection

with LCMV and vesicular stomatitis virus. We believe that the

different nature of the viruses plus the different virus/antigen

loads might play important roles for the expansion. In our previous

studies on LCMV infections in CD3gLLAA mice, we found that the

spleen virus titers were only slightly increased in CD3gLLAA mice

compared with wild-type mice [29]; however, in the present study

we find that the spleen virus titers are highly increased by 100- to

1000-fold on day 6 in CD3gLLAA compared with wild-type mice.

Thus, whereas the antigen load in CD3gLLAA and wild-type mice is

equivalent during LCMV infection, it is highly increased in

CD3gLLAA compared with wild-type mice during poxvirus infec-

tions, and we believe that this could be a significant factor in the

different numbers of virus-specific Tc cells found in CD3gLLAA and

wild-type mice during LCMV and poxvirus infection.

The significant role of the CD3g diL motif in T-cell biology is

supported by the observation that this motif has been conserved

for more than 350 million years of evolution and that it is even

found in the common ancestor of the CD3g and d chains in

amphibians [45]. In previous studies we have shown that the CD3gdiL motif fine-tunes the expansion of virus-specific Tc cells during

infections with LCMV and vesicular stomatitis virus [29], and that

the motif is involved in T-cell homeostasis [32]. However, mice

with a mutated CD3g diL motif did not show increased morbidity

to LCMV and vesicular stomatitis virus or succumbed earlier

because of a defective T-cell homeostasis. Thus, other more life-

threatening factors must have caused the evolutionary pressure

that has kept the CD3g diL motif intact for millions of years. We

believe that this study indicates that the role of the CD3g diL motif

in enhancing perforin/granzyme-mediated Tc cell cytotoxic by

�50% and thereby increasing the protection against acute, life-

threatening virus infections could have been a significant factor in

the preservation of the motif during vertebrata evolution.

Materials and methods

Mice and cell lines

Generation of the CD3gLLAA knock-in and P14LLAA mice strains

has been described previously [29]. C57BL/6, P14 [30, 31] and

gzmB–/– mice [34, 46] backcrossed to C57BL/6 were used as

controls. The transgenic Va2Vb8 TCR from P14 mouse is specific

for the LCMV glycoprotein gp33–41 epitope when bound to MHC

H-2Db [30]. Animal experiments performed in Denmark were

approved by the Animal Experiments Inspectorate, The Danish

Ministry of Justice (approval number 2007/561-1357). The ECTV

experiments were performed at the John Curtin School of

Medical Research, Canberra, in accordance with the Australian

National University Animal Experimentations Ethics Committee.

EL-4 cells were cultured in RPMI 1640 supplemented with

10% fetal calf serum, 1% L-glutamine, 0.5 IU/L penicillin and

500 mg/L streptomycin at 371C in 5% CO2. MEF and BSC-1 cells

were grown as previously described [47].

Expansion of TCR transgenic Tc cells

Splenocytes were isolated from TCR transgenic P14 and P14LLAA

mice and stimulated with 5 ng/mL of LCMV gp33–41 (Schafer-N)

for 3 days in complete medium (RPMI 1640 supplemented with

10% fetal calf serum, 1% L-glutamine, 0.5 IU/L penicillin,

500 mg/L streptomycin, and 50 mM 2-mercaptoethanol) in the

presence of 20 U/mL IL-2 at 371C in 5% CO2. The cells were

subsequently washed and rested for 2 days in complete medium

supplemented with 10 U/mL IL-2. The purity and the activation

state of the expanded TCR transgenic Tc cell population were

tested by flow cytometry after labeling with antibodies against

CD8a (clone 53-6.7), TCRVb8 (clone F23.1), TCRVa2 (clone

B20.1), and CD44 (clone IM7) from BD Biosciences; granzyme B

(clone GB11) from Invitrogen; and perforin (anti-Perforin-FITC

mAb, clone CB5.4) from Kamiya Biomedical Company.

51Cr release assay

EL-4 cells were used as target cells for the in vitro propagated P14

and P14LLAA Tc cells, and MEF cells were used for the in vivo

expanded Tc cells isolated 6 days post-infection with HE-ECTV.

Target cells were pulsed with 30mCi of Na2(51Cr)O4 (Perkin Elmer)

per 106 cells. EL-4 cells were either pulsed with gp33–41 or mock-

pulsed. MEF cells were infected with Mo-ECTV 5 PFU per cell (MOI

5:1). Tc and target cells were added to 96 round-bottomed plates at

the indicated effector-to-target cell ratios and co-cultured for 5 h at

371C in 5% CO2. The supernatants were subsequently analyzed and

the percentage of specific (51Cr) release was calculated as

((cpmexperimental release-cpmspontaneous release)/(cpmmaximum release-cpm

spontaneous release))� 100%. To distinguish the cytotoxicity mediated

by the perforin/granzyme and the Fas/FasL pathways, Tc cells were

in some experiments incubated with 100 nM concanamycin A

(Sigma-Aldrich) for 2 h before adding the target cells [33].

Cytokines and granzyme B production and secretion

To determine the ability of the Tc cells to produce cytokines, we

cultured the cells on gp33–41 pulsed splenocytes for the indicated



time at 371C in complete RPMI 1640 medium supplemented with

IL-2 and 3 mM monensin (Sigma-Aldrich) and subsequently

stained them with mAb against CD8, TCRVa2, CD44, and IFN-g(clone XMG1.2) or TNF-a (clone MP6.XT22) as previously

described [29, 32]. To determine the level of intracellular

granzyme B we stained the cells with mAb against CD8, TCRVa2,

CD44 and granzyme B (clone GB11). Cells were subsequently

analyzed by flow cytometry. Granzyme B, IFN-g and TNF-asecretion from activated Tc cells were measured by ELISA

according to the manufacturer (eBioscience).

Ectromelia virus infection

The Mo-ECTV and HE-ECTV strains were prepared and titrated as

described [48]. For the generation of virus-specific Tc cells

to be used in cytotoxicity assays, mice were infected with 1�104

PFU avirulent HE-ECTV i.v. For in vivo pathogenicity experi-

ments, mice were infected with 1�103 PFU virulent Mo-ECTV

s.c. and monitored for morbidity and mortality. Death was noted

when mice were sacrificed upon reaching a predefined clinical

endpoint score or when found dead. To determine virus

infectivity, spleens from mice infected for 4 or 6 days with

1� 103 PFU Mo-ECTV were weighed and processed to a 10%

lysate. The lysate was homogenized and frozen, and viral titers

were determined by plaque assay [34]. Virus infectivity was

expressed as log10 PFU/mL tissue lysate. A dominant epitope of

ECTV is B8R20–27 when presented by H-2Kb [49]. In some

experiments, the number of virus-specific Tc cells was determined

by staining with B8R20–27-Kb-tetramers (Ev-Kb-tet) and mAb

against CD8.

TCR down-regulation, degradation and Western blot

TCR down-regulation was measured by incubating P14

and P14LLAA Tc cells with 50 nM of the PKC activator phorbol

12,13-dibutyrate for the time indicated followed by the

determination of the TCR MFI by flow cytometry as previously

described [29]. TCR down-regulation was calculated as

((MFI of untreated cells minus MFI of treated cells)/MFI of

untreated cells)�100%. To determine TCR degradation,

P14 and P14LLAA Tc cells were washed once in complete

medium and once in PBS and resuspended in PBS with 0.5 mg/

mL sulfo-NHS-biotin (Pierce). The cells were incubated

for 30 min on ice, washed and resuspended in complete

medium to a concentration of 4�106 cells/mL. The biotinylated

cells were stimulated for 0, 2, 4, and 6 h with 1 mg/mL gp33–41 at

371C. Biotinylated TCR was subsequently determined and

quantitated as previously described [50]. Tyrosine-phosphory-

lated c-Cbl, total c-Cbl, Vav1, and GAPDH were determined by

Western blot using anti-c-Cbl-pY774 (]3555, Cell Signaling

Technology), anti-c-Cbl (clone 7G10, Upstate Biotechnology),

anti-Vav1 and anti-GAPDH (both from Abcam) as previously

described [29].

RNA interference

P14 and P14LLAA Tc cells were transfected with 100 ng siRNA

c-Cbl (]EMU070381, Sigma-Aldrich) with Amexa Nucleofector I

system at day 4 after peptide stimulation. A total of 3� 106 cells

were resuspended in 100mL of Amexa Mouse T cell Nucleofector

kit, mixed with siRNA, and immediately transfected with program

X-01. The cells were rested for 24 h at 371C and assayed as

indicated. The negative siRNA control was obtained from Ambion.

Statistical analysis

Statistical analyses were performed using Student’s t-test with a

5% significance level, unpaired observations, and equal variance.

For analysis of the survival data, we used the log-rank/

Mantel–Cox test.

Acknowledgements: The expert technical help of Aulikki

Koskinen and Bodil Nielsen is gratefully acknowledged. This

work was supported by grants from The Danish Medical Research

Council, The Novo Nordisk Foundation, The Lundbeck

Foundation, The A.P. Møller Foundation for the Advancement

of Medical Sciences, The Agnes and Poul Friis Foundation, The

Director Jacob Madsen & Wife Olga Madsens Foundation, and

The Astrid Thaysen Foundation for Basic Medical Sciences.

Conflict of interest: The authors declare no financial or

commercial conflict of interests.

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Abbreviations: diL: di-leucine-based � ECTV: ectromelia virus � Ev-Kb-

tet: B8R20–27-Kb-tetramer � FasL: Fas ligand � gzmB�/�: granzyme B

knock-out � HE-ECTV: Hampstead Egg ectromelia virus � LCMV:

lymphocytic choriomeningitis virus � MEF: mouse embryo fibroblasts

� Mo-ECTV: Moscow ectromelia virus � Tc: cytotoxic T cell

Full correspondence: Prof. Carsten Geisler, Department of International

Health, Immunology and Microbiology, Faculty of Health Sciences,

University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen,

Denmark

e-mail: [email protected]

Fax: 145-3532-7853

Received: 12/1/2011

Revised: 16/3/2011

Accepted: 21/4/2011

Accepted article online: 13/5/2011



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TCR down-regulation boosts T-cell-mediated cytotoxicity and protection against poxvirus infections

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