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INSERM U563, Centre de Physiopathologie de Toulouse Purpan, IFR 30, Centre Hospitalier Purpan, 31024 Toulouse Cedex, France
Correspondence
Jean-Luc Davignon
davignon{at}toulouse.inserm.fr
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ABSTRACT |
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) production was significant. Furthermore, cytotoxicity against peptide-pulsed targets was inhibited by HCMV infection, whereas IFN- production was not modified, suggesting that antigen processing was not altered. Remarkably, degranulation of CD4+ T cells in the presence of infected targets was significant. Together, our data suggest that impaired cytotoxicity is not due to failure to recognize infected targets but rather to a mechanism specifically related to cytotoxicity. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Cellular immunity plays a major role in the control of HCMV infection (Reddehase, 2000). CD8+ T lymphocytes are involved in this control through their cytotoxic function. Several reports illustrate the role of CD4+ T cells in the control of HCMV infection: (i) the lack of detectable anti-HCMV CD4+ T cells has been linked to HCMV-associated retinitis in human immunodeficiency virus (HIV) patients (Komanduri et al., 1998). (ii) More recently, Gamadia et al. (2003) found that impaired control of viral replication could be explained by the lack of gamma interferon (IFN-)-secreting CD4+ T cells. (iii) Persistence of infused HCMV-specific CD8+ T cells in patients depends on the presence of CD4+ T cells (Einsele et al., 2002; Walter et al., 1995). Mechanisms that account for the elimination of viruses by T lymphocytes can be divided into two main categories: release of cytokines and cytotoxicity.
There are two major pathways of T lymphocyte-mediated cytotoxicity described: the Fas and the perforin/granzyme pathways (Kagi et al., 1994). CD4+ T cells have been recently described as capable of killing specific target cells through the perforin/granzyme pathway (Appay, 2004). Moreover, circulating CD4+ T cells from HCMV seropositive donors have been shown to express perforin and granzyme ex vivo, suggesting that they are capable of performing cytotoxicity through this pathway (van Leeuwen et al., 2004). Although HCMV-specific CD4+ T cells have been shown to kill antigen (Ag)-pulsed targets (Hegde et al., 2005), it is, so far, unknown whether HCMV-specific CD4+ T cells are actually able to kill HCMV-infected targets.
In vivo, MHC II-expressing cells such as macrophages can be infected by HCMV (Bissinger et al., 2002; Sinzger et al., 1996). Although there is so far no formal proof that dendritic cells can be infected in vivo, those cells have been demonstrated to be permissive in vitro to so called endotheliotropic strains of HCMV (Sinzger et al., 2000). Thus evaluating whether infected MHC II-expressing cells are sensitive to cytotoxic CD4+ T cells is of crucial importance, especially considering the numerous mechanisms of escape of HCMV from the CD4+ T-cell response (Hegde et al., 2003).
Cell models have been developed to assess the function of CD4+ T lymphocytes on HCMV-infected antigen presenting cells (APCs). We and others have obtained cell lines that constitutively express MHC II and are permissive to HCMV in vitro (Cebulla et al., 2002; Le Roy et al., 1999; Tomazin et al., 1999). We have previously shown that HCMV-infected APCs can present endogenous IE1 protein to specific CD4+ T-cell clones whose activation results in the control of infection in vitro (Le Roy et al., 2002).
In this paper, we have analysed the cytotoxic activity of IE1-specific CD4+ T-cell clones against infected APCs used as targets. We found that although IE1-specific CD4+ T-cell clones could kill targets loaded with IE1 peptide, their cytotoxicity against HCMV-infected targets was not detected. This was not due to a defect in the amount of epitope provided by the infection since, similar to a previous paper from our laboratory (Le Roy et al., 2002), IFN- production by CD4+ T cells was abundant. Rather, cytotoxicity was specifically impaired at the effector, but not recognition, phase. Our data describe for the first time the inhibition of cytotoxicity by CD4+ T lymphocytes towards HCMV-infected MHC II-expressing targets.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Human astrocytoma cells U373MG were a kind gift from S. Michelson (formerly at Pasteur Institute, Paris, France). The EBV-transformed B lymphoblastoid cell line (Steinlin) was from the Xth Histocompatibility Workshop. Both were cultured in medium supplemented with 10 % fetal calf serum (FCS). U373MG-CIITA cells were obtained as described previously (Le Roy et al., 1999) by transfection of U373MG with the pSR
Neo/CIITA plasmid and were cloned by limiting dilution. U373MG-CIITA cells have been typed as HLA-DR3 (Le Roy et al., 1999).
IE1-specific, HLA-DR3-restricted FzD11, FzB1 and FzD3 CD4+ T-cell clones have been previously reported (Davignon et al., 1996). Cells were periodically restimulated in the presence of phytohaemagglutinin, IL-2 and allogeneic irradiated peripheral blood mononuclear cells and maintained in culture medium supplemented with 10 % AB human serum, as described previously (Davignon et al., 1996).
HCMV (Towne) stocks were obtained by infection of MRC5 cells (bioMérieux) at an m.o.i. of 0.1 in 10 % FCS culture medium. Towne strain virus titration was performed, as described previously (Le Roy et al., 2002), on MRC5 cells (Pasteur Mérieux).
Antibodies and reagents.
Blocking anti-Fas (CD95) monoclonal antibody (mAb) ZB4 was purchased from Upstate. Fluorescein isothiocyanate (FITC)-coupled anti-CD107a mAb was purchased from BD Biosciences. Phycoerythrin (PE)-Cy5-coupled mAb and isotype controls were purchased from eBioscience. Monensin was purchased from Sigma-Aldrich. Concanamycin A (CMA), an inhibitor of perforin-based cytotoxicity (Kataoka et al., 1996), was purchased from Sigma-Aldrich. Radioactive 51Cr-sodium chromate was purchased from MP Biochemicals. Synthetic peptides were purchased from the core facility at the University of Augusta (GA).
Cell staining.
After centrifugation, pellets of lymphocytes were fixed in 10 % buffered formalin in an Eppendorf tube and were embedded in a paraffin block. Two mAbs directed against granzyme B clone 5B10 (Novocastra) and perforin clone GrB7 (Dakocytomation) were used in parallel. Immunostaining was performed with a streptavidin-biotin three-stage technique, using the Strept ABC complex/HRP Duet kit (Dakocytomation). The signal was revealed with diaminobenzidine as a chromogen.
Antigen presentation by U373MG-CIITA and EBV-B cells, and measurement of CD4+ T-cell response.
U373MG-CIITA cells (105 per well) were plated in six-well plates. After 24 h, they were infected with HCMV (m.o.i. of 3) and/or pulsed with IE1 (91–110) or various concentrations of IE1 (88–101) peptide for 2 h and simultaneously labelled with 51Cr. Cells were then extensively washed to remove peptide, trypsinized, washed again, counted, transferred to round bottomed 96-well plates and incubated with the IE1-specific FzD11 CD4+ T-cell clone for 5 h. The same procedure was used for EBV-B cells except that they were pulsed (5x105 cells ml–1) with Ag in 24-well plates and subsequently seeded in 96-well plates. Spontaneous release was <25 % throughout. In both cases, supernatant was collected after 24 h of culture. Specific cytotoxicity was calculated as (experimental c.p.m. release–spontaneous c.p.m. release/total c.p.m. release–spontaneous c.p.m. release).
IFN- production by IE1-specific CD4+ T-cell clone was measured in supernatants in an ELISA by using a pair of IFN--specific mAbs from eBiosciences.
Measurement of CD4+ T-cell clone degranulation.
Acquisition of cell surface CD107a, as a marker of degranulation, by IE1-specific FzD11 CD4+ T-cell clone was performed according to the article by Betts et al. (2003). Briefly, U373MG-CIITA cells, used as targets, were infected with HCMV (m.o.i. of 3) for 4 days, or left uninfected, then incubated or not in the presence of IE1 (88–101) peptide for 2 h and extensively washed. U373MG-CIITA cells were then trypsinized and added to effector cells FzD11. Incubation was performed at 37 °C for 6 h in the presence of monensin (3 µM) and mAbs specific for CD4 and CD107a. Cells were then washed twice in PBS and fixed with 2 % paraformaldehyde, then analysed on a Coulter XL flow cytometer.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Hegde et al., 2005). First, stainings for perforin (Fig. 1a) and granzyme (Fig. 1b) expression established positivity of CD4+ T-cell clones for those markers. We then tested cytotoxicity of IE1-specific CD4+ T-cell clones in standard 5 h cytolysis assays on Steinlin LCL cells. CMA, a specific inhibitor of perforin-based cytotoxicity (Kataoka et al., 1996), inhibited cytotoxicity by IE1-specific CD4+ T-cell clones (Fig. 1c). By contrast, the anti-Fas antibody ZB4 did not inhibit cytotoxicity. However, it must be stressed that the duration of the assay was 5 h, which is typical of perforin-based cytotoxicity. Nevertheless, we demonstrated that Fas-based cytotoxicity was not involved in our assay, although it may occur over longer time of incubation. Altogether, these results indicate that perforin-based cytotoxicity was the main pathway of cytotoxicity used in those experiments.
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). As reported earlier for U373MG and U373MG-CIITA cells (Le Roy et al., 1999, 2002), 100 % of U373MG-CIITA cells were infected in the conditions used. To understand the respective requirements for cytotoxicity and IFN- production, we titrated cytotoxicity and IFN- responses to IE1 (88–101) peptide. This peptide was found to be the optimal 14-mer recognized by anti-IE1 CD4+ T-cell clones (data not shown). U373MG-CIITA cells were sensitized with various concentrations of peptide and cytotoxicity and IFN- production were evaluated in the same assay. To do so, supernatants were harvested from the cytotoxicity assay and tested for IFN- secretion. Data presented in Fig. 2 show IFN- response (Fig. 2a and c) as well as cytotoxicity response (Fig. 2b and d) in the same assay. Cytotoxicity seemed more sensitive than IFN- production in response to peptide since the median effective concentration (EC50) was 
0.01 nM for cytotoxicity, whereas it was >10 nM for IFN- production, although the absence of a plateau of IFN- production did not allow us to precisely calculate it. We then quantified what equivalent peptide concentration was produced by HCMV infection in our U373MG-CIITA test cells (Fig. 2a and c). The assay was performed on days 2, 3 and 4 post-infection (p.i.) (Fig. 2a for FzD11) and on day 2 p.i. with an m.o.i. of 10 (Fig. 2c for FzB1). The equivalent peptide reached 0.1 nM concentration in both cases. No cytotoxicity was observed in response to infected U373MG-CIITA cells whatever time p.i. (days 2, 3 or 4 p.i.) and at an m.o.i. of 3 (Fig. 2b) or 10 (Fig. 2d). Experiments performed with another CD4+ T-cell clone FzD3 also showed no response against infected targets despite IFN- response, although no titration of the response was evaluated. Thus peptide and infection were both capable of sensitizing CD4+ T cells for IFN- production, whereas only peptide allowed CD4+ T cells to kill target cells.
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production could be observed in those assays and since the threshold of peptide concentration seemed lower for cytotoxicity than for IFN- production (0.01 nM vs >10 nM, respectively), we envisaged that infection may cause inhibition of perforin-based cytotoxicity against peptide-pulsed targets.
Infection inhibited FzD11 CD4+ T-cell-mediated cytotoxicity on IE1 peptide-pulsed targets
To test the hypothesis that infection may inhibit perforin-based cytotoxicity, we performed an experiment in which we measured the levels of IFN- produced by CD4+ T cells in the presence of peptide-pulsed APC, infected or uninfected, on the one hand, and cytotoxicity on the other hand. To do so, similar to Fig. 2
, supernatants were harvested from the cytotoxicity assay and tested for IFN- secretion. Fig. 3 illustrates that the effector : target ratios used in all experiments clearly fall into the linear range of cytotoxic response, which confirmed and extended the data of Fig. 2. Again, in the presence of infected cells, the cytotoxicity was negligible (2 %), while IFN- production was high (1800 pg ml–1). The level of cytotoxicity observed in the presence of peptide-pulsed-infected targets at a 20 : 1 effector : target ratio was lower than that observed at a 5 : 1 ratio in the presence of peptide-pulsed-uninfected targets, demonstrating that there was at least a fourfold decrease in the intensity of cytotoxicity when targets were infected. However, in the same conditions, the levels of IFN- production were equivalent, suggesting that the inhibition was targeted to cytotoxicity but not to IFN- production.
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). We confirmed that degranulation of CD4+ T cells did occur in response to targets incubated with peptide and showed that this response was correlated with the amount of peptide (Fig. 4a). Percentage of CD107a-positive CD4+ cells was evaluated from samples depicted in Fig. 4(a) and plotted in Fig. 4(b). Degranulation in response to infected targets could be titrated to an equivalent peptide of 0.1 nM (Fig. 4b), which was equal to IFN- production in response to infected targets (Fig. 2). CD107a stainings performed in duplicate gave highly reproducible results as responses were 76.6 and 76.9 % against 1000 nM peptide, 15.1 and 15.2 % against HCMV, and 2.6 and 3 % in the absence of Ag (No Ag). In addition, contrary to the observation in Fig. 3 in a 51Cr-release assay, infection did not impair degranulation of CD4+ T cells incubated in the presence of targets pulsed with peptide (Fig. 4c). Thus, inhibition of cytotoxicity observed in the 51Cr-release assay did not appear to be due to inhibition of CD4+ cytotoxic function but rather to resistance of infected targets, although further investigation is required to define the precise mechanism.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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CD4+ T cells have been demonstrated recently to express perforin and granzyme and to be able to kill through the perforin/granzyme pathway (Appay, 2004). CD4+ T cells expressing granzyme B have been found ex vivo (Appay et al., 2002; van Leeuwen et al., 2004) and have been suggested as potential actors of the elimination of viruses. The present study shows that the pathway used by cytotoxic IE1-specific CD4+ T lymphocytes against targets sensitized with IE1 peptides was perforin based, although use of the granzyme pathway was not formally proven. Although cloned CD4+ T cells may acquire perforin/granzyme expression over prolonged time of culture, it appears that perforin/granzyme-positive CD4+ T cells belong to a highly differentiated population that can be found ex vivo (Appay, 2004; Appay et al., 2002).
Cytotoxity against peptide-loaded targets seems to require less peptide than IFN- production. This had already been reported for CD8+ T cells (Betts et al., 2004; Valitutti et al., 1996) but was controversial regarding CD4+ T cells (Hemmer et al., 1998; Valitutti et al., 1996). However, production of other cytokines by CD4+ T cells may not obey the same rule as demonstrated for IL-4 production (Hemmer et al., 1998). Another finding of the present study was the equivalent peptide concentration obtained in HCMV-infected U373MG-CIITA cells. This had not been previously determined for the CD4+ T-cell response against HCMV. This concentration was calculated from the IFN- response and should have been sufficient to induce a significant cytotoxic response. It appeared from our experiments that the sensitivity of the CD4+ T-cell cytotoxic response was lower than that usually found with CD8+ T cells (Gavin et al., 1993; Valitutti et al., 1996). This suggests that CD8+ T cells are more potent than CD4+ T cells with regard to cytotoxicity, although this requires a more thorough analysis of the two responses against the same pathogen in the same individual. This functional difference may explain why CD8 cytotoxicity can be observed on infected targets at relatively low m.o.i. (Warren et al., 1994). However, because of the multiple kinetics of escape of HCMV from CD4+ responses on the one hand (Cebulla et al., 2002; Miller et al., 2002) and CD8+ T-cell responses on the other hand (Johnson & Hill, 1998), comparison of cytotoxicity between CD8+ and CD4+ T cells on infected cells may prove difficult. It is noteworthy that, similar to this present paper on CD4+ T cells, some anti-HIV1 CD8+ T cells have been reported to secrete IFN- in the absence of detectable cytotoxicity, although degranulation was not investigated (Horton et al., 2004).
HCMV-specific cytotoxic CD4+ T cells have been previously described. However, targets consisted of cells infected with recombinant vaccine (Hopkins et al., 1996), adenoviruses (Hegde et al., 2005) or canarypox viruses (Gyulai et al., 2000), or pulsed with soluble Ag (Tazume et al., 2004) or peptides (Elkington & Khanna, 2005; Gautier et al., 1996; Tazume et al., 2004; Weekes et al., 2004; Zaunders et al., 2004). Anti-HCMV CD4+ cytotoxicity has been previously reported to depend on perforin/granzyme pathways (Tazume et al., 2004). In that study, cytotoxicity was tested on infected fibroblasts but it required longer time of incubation (16 h) and was partially dependent on Fas (Tazume et al., 2004). In addition, fibroblasts do not spontaneously express MHC II. Thus, up to now, the lack of study on MHC II-positive infected targets may have been due to the relative unavailability of defined permissive APCs. This is the first study to evaluate this point using clonal HCMV-specific CD4+ T cells. Other specificities will have to be tested to confirm that this observation can be extended to various protein targets in addition to IE1. Although this study was performed with a limited number of clones displaying the same specificity, it appears from two different laboratories that the cytotoxic CD4+ T-cell response against HCMV proteins evaluated using peptides is extremely focused against a single epitope, reflecting clonal selection (F. Kern and P. Moss, personal communications). Thus our data may actually reflect broader anti-IE1 responses.
U373MG cells have been extensively used in vitro and are considered as fully permissive to HCMV (Cebulla et al., 2002; Hegde et al., 2005; Le Roy et al., 1999, 2002). Transfection of CIITA has allowed for expression of HLA class II molecules by U373MG cells and has been used in independent laboratories to study either presentation of HCMV antigens (Hegde et al., 2005; Le Roy et al., 2002; Tomazin et al., 1999) or downregulation of HLA-DR by HCMV infection (Cebulla et al., 2002; Le Roy et al., 1999; Tomazin et al., 1999). We have found repeatedly that CD4+ T-cell recognition of infected U373MG-CIITA cells is significant with regard to IFN- production (Le Roy et al., 1999, 2002). In our hands, inhibition of HLA-DR expression in U373MG-CIITA cells always occurred after 5 days of infection (data not shown). In the present study, IFN- response was again clearly observed and inhibition of response was specifically targeted towards cytotoxicity. Whatever consequences inhibition of MHC II expression would have on CD4+ T-cell responses, it should have applied to both IFN- secretion and cytotoxicity. It thus appears that our study does not point to a defect in the mechanism of Ag processing or recognition but instead is related to the effector phase of the cytotoxic response. In a previous report, we showed that anti-IE1 CD4+ T cells were able to kill infected APCs (Le Roy et al., 2002). However, this occurred in a long-term assay, over several days of culture, and was different from the assays presented here.
The mechanism through which cytotoxicity is inhibited has yet to be discovered, but our data are consistent with resistance of targets cells to effector mechanisms such as degranulation. Several potential mechanisms may be envisaged. Inhibitor ligands could be induced by HCMV on target cells and could prevent cytotoxicity without affecting degranulation. For example, inhibitors of serine protease inhibitors (serpin) proteases have been reported to protect immature dendritic cells (Medema et al., 2001b) and tumour cells (Medema et al., 2001a) from cytotoxicity. HCMV-encoded pUL16 has been shown to protect against NK-mediated lysis (Odeberg et al., 2003; Vales-Gomez et al., 2003).
CD4+ T-cell cytotoxic responses against virus-infected targets have been reported in herpes simplex virus (HSV) (Yasukawa et al., 1996), polio (Wahid et al., 2005) and Epstein–Barr virus (EBV) (Adhikary et al., 2006; Khanolkar et al., 2001). It thus appears that HCMV, unlike other herpesviruses such as HSV1 and EBV, is able to inhibit the cytotoxic CD4+ T-cell response. Although other viruses have been demonstrated to escape from the immune system (Xu et al., 2001), HCMV has been described to devote a great part of its genome to the escape from the immune system (Mocarski, 2004). This suggests that, besides inducing one of the most intense immune reactions of all pathogens (Sylwester et al., 2005), HCMV has also evolved to keep a balance between the intensity of the host's response and its own capacity to resist the immune system. The demonstration of endogenous presentation of IE1 by the MHC II of infected cells (Le Roy et al., 2002) suggests an effective role of the mechanisms of escape from the CD4+ T-cell response because those mechanisms are anticipated to also address infected, IE1 expressing, targets. Inhibition of cytotoxicity by HCMV suggests that CD4+ T cells may participate in immunopathology by killing only uninfected targets acquiring soluble Ag. Since several cell types, including glial and epithelial cells (Fierz et al., 1985; Londei et al., 1984), can be induced to express MHC II in response to IFN-, presentation of soluble HCMV Ags may be a consequence of local immune responses. It may therefore be of interest to test HCMV-specific granzyme-positive CD4+ T cells found in the blood of HCMV seropositive patients (van Leeuwen et al., 2004) for their cytotoxic function on infected targets in comparison to uninfected, Ag-pulsed, targets.
In conclusion, we have described cytotoxicity of anti-IE1 CD4+ T cells against peptide-pulsed targets through the perforin pathway and observed that cytotoxicity required less Ag than IFN- production. We have also described an inhibition of cytotoxicity of anti-IE1 CD4+ T cells on HCMV-infected targets, whereas IFN- production was significant. Although data point to a mechanism of resistance of infected targets, further studies are required to precisely define the molecular basis for the inhibition. Our findings suggest that perforin/granzyme-mediated cytotoxicity of anti-HCMV CD4+ T cells against infected APCs may be limited and we need to extend further our knowledge of the recognition of HCMV-infected APCs by specific CD4+ T cells.
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ACKNOWLEDGEMENTS |
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Received 28 February 2007;
accepted 11 May 2007.
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