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The Journal of Immunology, 2005, 175: 6812-6819.
Copyright © 2005 by The American Association of Immunologists

Optimization of CD4+ T Lymphocyte Response to Human Cytomegalovirus Nuclear IE1 Protein through Modifications of Both Size and Cellular Localization1

Sandra Delmas*, Laurence Martin2,{dagger}, Michel Baron*, Jay A. Nelson{dagger}, Daniel N. Streblow{dagger} and Jean-Luc Davignon3,*

* Institut National de la Santé et de la Recherche Médicale Unité 563, Centre de Physiopathologie de Toulouse Purpan, Toulouse, France; and {dagger} Department of Molecular Microbiology and Immunology and The Vaccine and Gene Therapy Institute, Oregon Health Sciences University, Portland, OR 97239


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We have previously reported that the CD4+ T lymphocyte response against nuclear human CMV IE1 protein depends in part on endogenous MHC class II presentation. To optimize presentation by HLA-DR of the nuclear IE1 protein and increase the response by CD4+ T cells, we have constructed two different adenovirus vectors containing mutant versions of IE1, containing a HLA-DR3 epitope, fused to GFP. The first construct consisted of a sequence of 46 aa encoded by exon 4, called GFP-IE1 (86–131). The second construct consisted of the whole IE1 mutated on exon 4 nuclear localization signals, identified in this study, and deleted of already known exon 2 nuclear localization signals (GFP-IE1M). Both of these IE1 vectors expressed proteins with cytoplasmic localization, as evidenced by GFP expression, as opposed to control GFP-IE1, which was nuclear. GFP-IE1 (86–131) induced IE1-specific CD4+ T cell clone response that was >30-fold more potent than that against GFP-IE1 and GFP-IE1M. The CD4+ T cell response was due to endogenous presentation followed by exogenous presentation at later time points. Presentation was dependent on both proteasome and acidic compartments. GFP-IE1 (86–131) was rapidly degraded by the APC, which may account for better presentation. Our data show potentiation of the CD4+ T cell response to a specific epitope through shortening and relocation of an otherwise nuclear protein and suggest applications in vaccination.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human CMV (HCMV)4 is generally characterized by asymptomatic primary infections followed by subsequent lifelong latency. The main pathological consequences of HCMV are seen in congenital infections and following immunosuppression, either iatrogenic (transplantation, cancer) or infectious (AIDS) (1). HCMV remains a leading cause of birth defects and of pathology in immunosuppressed patients (1). A vaccine to HCMV is warranted based on the morbidity and mortality inflicted in these populations of virus-infected individuals.

The development of an effective HCMV vaccine relies on the understanding of the immune response toward the virus and on manipulations that may potentiate this immune response. Progress has been made in recent years in the identification both of HCMV-derived Ags recognized by the immune system and of the viral mechanisms used to escape immune surveillance (2, 3, 4). Multiple strategies have been suggested for vaccination against HCMV, which have been directed toward both cell-mediated and humoral immune responses (5). HCMV potentially encodes ~200 gene products, and transcription of these genes occurs in three kinetic phases designated as immediate early (IE), early (E), and late (L) (6). The relationships between viral genes transcription, localization, function, and the promotion of humoral and cell-mediated immune responses is unclear. Envelope glycoproteins such as gB, which are expressed during the late kinetic phase of virus replication, have been described as potential vaccines targeting the Ab response (7). The immediate early protein IE1 and the major tegument protein pp65 (early/late kinetics) have been described as targets for both CD4 and CD8 lymphocyte responses (8, 9, 10, 11, 12, 13, 14). We have recently reported that IE1 can be endogenously presented to CD4+ T lymphocytes, although the pathway of this presentation has not been identified (15). CD4+ T cells specific for nuclear IE1 were able to control infection in vitro through recognition of a specific HLA-DR3-restricted epitope (15). This appears to be of importance because the only other reported viral nucleoprotein endogenously presented by MHC class II (MHC II) molecules is the EBV-encoded protein nuclear Ag 1 (EBNA1) (16). However, little is known about the efficiency of Ag presentation and recognition of epitopes that are derived from nuclear vs nonnuclear cellular sites.

Nuclear targeting of proteins occurs through the recognition of specific nuclear localization signals (NLS), which are composed of basic amino acid residues (17). Nuclear proteins have been described to bear different patterns of NLS: those can be either unique, double (i.e., independent NLS located in different portions of the protein), or bipartite (a single NLS composed of two to six basic residues separated by several amino acids) (17). To better understand the presentation of IE1 and to test whether the nuclear localization of IE1 is crucial for its presentation by APC to specific CD4+ T cells, we have generated a series of truncated IE1 proteins tagged with the GFP. To date, only one NLS, encoded by exon 2, has been described in IE1 (18). By making a panel of IE1 mutations, we have identified a new NLS encoded within exon 4. We have used recombinant adenoviruses containing wild-type and mutant versions of IE1-GFP in combination with our previously described model of HLA-DR3-expressing APC (15) to analyze effects of cellular distribution in the presentation of HLA-DR3-restricted epitope using a specific CD4+ T cell clone. We found that the localization (nuclear vs cytoplasmic) had no quantitative influence on presentation but that the size of the protein influenced the intensity of the response. These findings have implications in the understanding of the presentation of nuclear proteins and the development of a HCMV vaccine.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells, Abs, reagents

U373MG cells were obtained from American Type Culture Collection. U373MG-CIITA cells have been described previously (15). Anti-GFP mAb 3E6 was obtained from Qbiogene. MG132 was purchased from Calbiochem, and chloroquine was purchased from Sigma-Aldrich. The HLA-DR3-restricted, IE1-specific CD4+ T cell clone, FzD11, has been described previously (15).

Plasmid constructions and transfections

The IE1 cDNA was cloned in pEGFPC1 (BD Clontech), downstream of the enhanced GFP (EGFP) gene. The recombinant plasmid was named pEGFP-IE1. Truncated IE1 cDNA sequences were obtained from pEGFP-IE1 as follows (see Fig. 1): pEGFP-{Delta}ES (deletion from residues 132 to 491) was obtained from pEGFPC1-IE1 using an EcoRV/SmaI cleavage. pEGFPC1 (residues 86–131) was obtained through BamHI and EcoRV digestions of pGEX-E4, described in a previous report (19). The resulting protein was named IE1 (86–131). pEGFPC1-e4 (residues 86–491 encoded by exon 4) was obtained from pGEX-E4 through digestions by BamHI and EcoRI. pEGFP-IE1M was obtained through XhoI and Bsp120L digestions of pEGFPIE1-dNLS2 to delete NLS1. After a Klenow reaction and the closure with a T4 DNA ligase of the truncated plasmid, pEGFP-IE1M was obtained.



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  		FIGURE 1. Summary of localization of truncated IE1 proteins in transfected U373MG cells. U373MG cells were transfected with plasmids encoding for various truncated GFP-IE1 proteins. Localization nucleus (N) vs whole cell (C) is indicated. NLS encoded by exon 4 was found to have the following sequence: 326-KRPLITKPEVISVMKRR-342. The IE1 (86–131) construction included the V-86-110-Q HLA-DR3-restricted epitope (17 ).

 
Cloning of the NLS encoded by exon 4 of IE1 (K-326-342-R) was performed by PCR using the following primers: forward, 5'-ATA CTC GAG CCA AGC GGC CTC TGA TAA CC-3', and reverse, 5'-ATA GGA TCC TTC ATG CAG ATC TCC TCA ATG-3', and cloning of IE1 (K-184-210-R) was performed using the following primers: forward, 5'-ATA CTC GAG CTA ACA AGT TGG GGG GTG CAG-3', and reverse, 5'-ATA GGA TCC TGT AGC ACA TAT ACA TCA TC-3' using the XhoI and BamHI sites of the pEGFPC1 plasmid.

Cloning of AAA-mutated NLS was performed by overlapping PCR using the following primers for the first PCR: forward ({Delta}NLS), 5'-ATC AGT GTA ATG GCG GCA GCT ATT GAG GAG ATC TGC ATG AAG GTC-3'; reverse (IE1), 5'-ATA TCT AGA TTA CTG GTC AGC CTT GCT TCT AGT CAC-3' (containing the XbaI site), then the following primers for the second PCR: forward (IE1), 5'-ATA GAA TTC ATG GAG TCC TCT GCC AAG AGA AAG-3' (containing the EcoRI site), and reverse ({Delta}NLS), 5'-GAT CTC CTC AAT AGC TGC CGC CAT TAC ACT GAT AAC CTC AGG C-3'. Then forward (IE1) and reverse (IE1) primers were used on the two PCR products for the overlapping PCR to generate the AAA-mutated NLS. Insertion of the amplified fragment into the pEGFPC1 plasmid was performed using the EcoRI and XbaI sites.

Transfections were performed using Fugene reagents (Roche), according to the instructions of the manufacturer. Transient transfections were examined under a fluorescence microscope after 48 h of culture.

Recombinant adenoviruses

Adenovirus vectors expressing GFP-IE1, GFP-IE1 (86–131), or GFP-IE1M were constructed by subcloning the DNA fragment into pAdTet7 using the restriction enzymes EcoRI and BamHI. pAd-Tet7 vector contains the tet-responsive enhancer within a minimal CMV promoter followed by the SV40 late poly(A) cassette, adenovirus E1A, and a single loxP site to increase recombination frequency. Recombinant adenoviruses were produced by cotransfection of 293 cells expressing the Cre recombinase with adenovirus DNA (Ad5-{psi}5) that contains an E1A/E3-deleted adenovirus genome and pAdTet7-GFP-IE1, pAdTet7-GFP-IE1 (86–131), or pAdTet7-GFP-IE1M construct (20). Recombinant adenoviruses were expanded on 293-Cre cells, and the bulk stocks were titered on 293 cells by limiting dilution in 96-well plates. IE expression was driven by coinfection with Ad-Trans expressing the Tet-off transactivator as described previously (21).

Endogenous Ag presentation by U373MG-CIITA cells

U373MG-CIITA were seeded (10,000 cells/well) and cultured in round-bottom 96-well-plates for 24 h. U373MG-CIITA were then incubated with different multiplicity of infection (MOI) of adenovirus expressing GFP-IE1 (Ad-GFP-IE1), GFP-IE1 (86–131) (Ad-GFP-IE1 86–131), GFP-IE1M (Ad-GFP-IE1M), and Ad-Tet-Trans (MOI 20). Cells were fixed at different times postinfection with 0.05% glutaraldehyde for 1 min, washed three times with PBS, and incubated with IE1-specific CD4+ T cell clone FzD11 clone (40,000 cells/well) as described above. Supernatants were collected after 24 h of culture.

Exogenous Ag presentation by U373MG-CIITA cells

Supernatants from U373MG-CIITA cells infected with adenovirus expressing GFP-IE1 (Ad-GFP-IE1), GFP-IE1 (86–131), Ad-GFP-IE1 (86–131), GFP-IE1M (Ad-GFP-IE1M), and Ad-Tet-Trans (MOI 20) were collected at different times postinfection. U373MG-CIITA cells were seeded (10,000 cells/well) and cultured in flat-bottom 96-well plates for 24 h and infected with these supernatants. After 24 h of infection, cells were fixed, washed three times, and incubated with IE1-specific CD4+ T cell clone FzD11 (40,000 cells/well) as described above. Supernatants were collected after 24 h of culture.

Inhibition of presentation

U373MG-CIITA were seeded (10,000 cells/well) and cultured for 24 h in flat-bottom 96-well plates. U373MG-CIITA cells were incubated with different MOI of adenovirus expressing GFP-IE1 (Ad-GFP-IE1), GFP-IE1 (86–131) (Ad-GFP-IE1 86–131), GFP-IE1M (Ad-GFP-IE1M), and Ad-Tet-Trans (MOI 20) for 3 h. Cells were washed three times with PBS and incubated with Chloroquine (80 µM) or MG132 (20 µM) for 24 h. Cells were then fixed, washed three times with PBS, and incubated with IE1-specific CD4+ T cell clone FzB11 clone (40,000 cells/well) as described above. Supernatants were collected after 24 h of culture. Control peptides IE1 (88–101) was used to ensure that inhibitors affect only Ag processing and not class II maturation or cell surface expression. Control cells were incubated with inhibitors only.

Tests of IE1-specific CD4+ T cells clone activation

IFN-{gamma} production by IE1-specific CD4+ T cell clone was measured in supernatants in an ELISA test. After collecting supernatants, wells were incubated with [3H]thymidine (1 µCi/well) for another 24 h. Proliferation was measured by evaluation of [3H]thymidine incorporation using a beta counter (Hewlett Packard).

Immunoprecipitation

U373MG-CIITA cells (2 x 105/well) were cultured in 6-well plates for 24 h. Cells were then infected during 16 h by Ad-GFP-IE1 (86–131) or Ad-GFP-IE1 at high MOI (1000) and Ad-Trans at MOI = 20. Cells were then deprived of cysteine and methionine in selective RPMI 1640 medium (RPMI Select-amine kit; Invitrogen Life Technologies) supplemented with 5% FCS for 1.5 h and pulsed with Easy Tag Express-35S protein labeling mix (NEN) at 200 µCi/ml of culture for another 1 h. Chase experiments were performed by replacing selective medium by complete 10% FCS-RPMI. At the indicated times, cells were scraped and lysed in buffer containing Tris 20 mM (pH 8.3), NaCl 50 mM, 1% Nonidet P-40, EDTA 1 mM, and inhibitors mixture 1/50 (P-8340; Sigma-Aldrich). Cells lysates were precleared with 20 µl of Zyzorbine (Zymed Laboratories) at 4°C for 30 min. After centrifugation, 1 µg of anti-GFP mAb 3E6 was added to the supernatant, and incubation was continued for 2 h, followed by the addition of 20 µl of protein G (Sigma-Aldrich) and a final incubation for 16 h at 4°C. After five washes with TBS (1 ml), the immunoprecipitates were denatured by boiling in sample buffer and loaded for electrophoresis on a 10% polyacrylamide/SDS gel.

Confocal microscopy

U373MG-CIITA cells were seeded and cultured on glass coverslips into 4-well plates (Nunc) at the density of 1.5 x 105 cells/well for 24 h. Cells were infected with adenovirus expressing GFP-IE1 (Ad-GFP-IE1), GFP-IE1 (86–131) (Ad-GFP-IE1 86–131), GFP-IE1M (Ad-GFP-IE1M) (MOI 60), and Ad-Tet-Trans (MOI 20) for 24 h. Cells were washed twice with PBS, fixed in PBS with 3% paraformaldehyde for 20 min, and washed twice. Cells were visualized using a Zeiss LSM 510 confocal microscope (Zeiss). Excitation wavelength = 488 nm, emission wavelength = 509 nm; objective = plan-Apochromat x63/1.4 oil.

Flow cytometry

U373MG-CIITA cells were infected with adenovirus expressing GFP-IE1, GFP-IE1 (86–131), and GFP-IE1M for 48 h. Cells were then incubated with MG132 (20 µM) or chloroquine (80 µM) for the last 20 h, detached with trypsin, fixed with 4% paraformaldehyde, and examined by flow cytometry for GFP fluorescence intensity using a Coulter XL flow cytometer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Identification of a new IE1-NLS

Because IE1 is a nuclear protein but is nevertheless endogenously presented by MHC II molecules, we questioned whether modifying the cellular localization of a portion of IE1 comprising the HLA-DR3-restricted epitope would enhance the CD4+ T cell response. To alter the nuclear localization of IE1, we first sought to identify the NLS motifs of IE1 with the goal to introduce modifications in the protein sequence. Using the computer program MacVector, we analyzed the IE1 primary sequence to identify potential NLS basic amino acid sequences and identified two potential bipartite NLS within IE1. These two NLS were then cloned in transductional fusion with GFP. GFP-IE1 (K-184-210-R) and GFP-IE1 (K-326-342-R) synthesis and localization were observed in transitory transfectants of U373MG cells. As shown in Fig. 1, only IE1 (K-326-342-R) was a genuine NLS because GFP-IE1 (K-326-342-R), but not GFP-IE1 (K-184-210-R), was routed to the nucleus. Therefore, we identified the IE1 (K-326-342-R) sequence as a new NLS in IE1. Because a NLS had been reported in exon 2 (18), we made another construction, IE1 {Delta}ES, encompassing exons 2 + 3 and a short (46 aa) sequence encoded by exon 4. This construction, allowing for the routing of GFP into the nucleus, confirmed the presence of a NLS in that region, although it did not focus specifically on exon 2 alone. The two NLS, one encoded by exon 2 (18) and the other by exon 4, as demonstrated in this work, could independently induce the routage of GFP to the nucleus as revealed by constructions NLS K-326-342-R and IE1 {Delta}ES. Mutating NLS K-326-342-R by Ala at positions 340–342 of the complete IE1 gene did not influence the location of GFP, possibly reflecting the nuclear routing of GFP by the NLS encoded by exon 2.

Potentiation of CD4+ T cell response against IE1

To alter the intracellular location of the HLA-DR3 epitope of IE1, a 47-aa sequence encoded by exon 4 was produced in fusion with GFP and named IE1 (86–131). This induced the cytoplasmic location of a truncated protein, devoid of NLS1 (encoded by exon 2) and NLS2 (K-326-342-R, encoded by exon 4) and containing the previously described HLA-DR3 epitope V-86-110-Q (22) in fusion with GFP (Fig. 1). Another construction named IE1M was obtained by deletion of exon 2 (24 aa) containing NLS1 and mutation of NLS2 (Ala, Ala, Ala mutation). Although IE1M was similar in size compared with the wild-type IE1 protein, the resulting protein was cytoplasmic (Fig. 1).

To identify effects that cellular distribution and size have on IE1 immune recognition, we produced adenoviruses recombinant for GFP-IE1 GFP-IE1M, and GFP-IE1 (86–131). Confocal microscopy analysis of U373MG-CIITA cells infected with adenovirus expressing GFP-IE1, GPF-IE1 (86–131), and GFP-IE1M was performed to determine the localization of the different proteins in our system. As anticipated, expression of GFP-IE1 protein was observed only within the nucleus (Fig. 2), whereas GFP-IE1M, in which NLS1 was deleted and NLS2 was mutated, was found in the cytoplasm (Fig. 2). The shorter protein GFP-IE1 (86–131) was also found in the cytoplasm (Fig. 2). These results matched those observed in transfection of U373MG cells, which are summarized in Fig. 1.



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  		FIGURE 2. Localization of proteins produced after infection of U373MG-CIITA with adenoviruses expressing variants of the IE1 protein. Adenoviruses expressing GFP-IE1, GFP-IE1 (86–131), and GFP-IE1M (MOI 60) together with Ad-Tet-Trans (MOI 20) were used to infect U373MG-CIITA cells. Localization of GFP was examined by confocal microscopy 24 h p.i. Original magnification, x63.

 
We then determined whether the cellular distribution of IE1 influenced the processing and presentation of the HLA-DR3-epitope. For this purpose, U373MG-CIITA cells were infected with adenovirus expressing GFP-IE1, GFP-IE1M, and GFP-IE1 (86–131) at different MOI in the presence of Ad-Tet-Trans. Fig. 3 shows activation of the FzD11 T cell clone in response to presentation of the different proteins by U373MG-CIITA. Proliferation (Fig. 3A) and production of IFN-{gamma} (Fig. 3B) were evaluated.



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  		FIGURE 3. Potentiation of CD4+ T cell response against IE1. U373MG-CIITA cells, used as APC, were infected with Ad-GFP-IE1 ({diamondsuit}), Ad-GFP-IE1 (86–131) ({blacksquare}), and Ad-GFP-IE1M ({blacktriangleup}) at different MOI together with Ad-Tet-Trans (MOI 20). U373MG-CIITA cells infected with Ad-GFP-IE1, Ad-GFP-IE1 (86–131), and Ad-GFP-IE1M but not with Ad-Tet-Trans were used as negative controls (control AdenoV). U373MG-CIITA cells incubated in the presence of IE1 peptide (88–101) (•) or in the absence of peptide ({circ}) were used as positive and negative controls, respectively. Proliferation of CD4+ T cell clone FzD11 in the presence of infected U373MG-CIITA was measured in a [3H]thymidine assay (A) and IFN-{gamma} production assay (B) after various times of infection.

 
As shown in Fig. 3A, at 24 h postinfection (p.i.), proliferation of CD4+ T cell clone FzD11 was >10- to 30-fold higher using U373MG-CIITA infected with Ad-GFP-IE1 (86–131) compared with Ad-GFP-IE1 and Ad-GFP-IE1M: similar proliferation required a 10- to 30-fold more Ad-GFP-IE1 and Ad-GFP-IE1M. On day 2 p.i., proliferation of FzD11 T cell clone was increased compared with 24 h p.i. Proliferation in response to GFP-IE1 (86–131) protein was higher than at day 1 p.i., and the difference with the two other proteins was even greater and reached a >30-fold potentiation. On day 3, the responses to the three proteins were equivalent. The production of IFN-{gamma} by the T cell clone when cultured with adenovirus infected U373MG-CIITA paralleled the proliferation data described above, although the differences between the IE1 mutants were somewhat less pronounced (Fig. 3B). Because IE1 has been shown to be released in the culture medium due to viral cytopathic effects (15) and to investigate whether proteins with different localizations would be presented from different pathways, supernatants from the experiment shown in Fig. 3A (before the addition of T cell clone) were collected and used to sensitize fresh U373MG-CIITA cells, and the response of FzD11 T cell clone was analyzed. Data in Fig. 4A showed that after 24 h of infection, all the proliferation was due to endogenous presentation because no response was observed using the supernatant as the source of released Ag (except a modest response to GFP-IE1 at the highest MOI). On day 2 p.i., although the response to GFP-IE1 remained mostly endogenous, most of the responses to the cytoplasmic proteins, GFP-IEM and GFP-IE1 (86–131), were from exogenous presentation. On day 3 p.i., the response to nuclear GFP-IE1, as well as to the two other proteins, resulted from exogenous sources, similar to what is observed in HCMV infection after longer times of infection (15). The IFN-{gamma} response (Fig. 4B) paralleled that of proliferation (Fig. 4A): little or no IFN-{gamma} was produced after incubation with 24-h supernatants. Again, data from days 2 and 3 p.i. showed that IFN-{gamma} production by FzD11 T cell clone to nuclear GFP-IE1 was due mostly to endogenous presentation. In conclusion, the presentation of an epitope derived from nuclear protein GFP-IE1 was endogenous. Modifying the routage and size of IE1, as in GFP-IE1 (86–131), improved the CD4+ T cell response. The presentation of this cytoplasmic protein, endogenous at early time points, became rapidly from exogenous sources. Modifying the localization but not the size (GFP-IE1M) did not significantly improve the overall CD4+ T cell response but changed the mode of presentation, which occurred mostly via the exogenous processing/presentation pathway.



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  		FIGURE 4. Presentation of exogenous and endogenous IE1 by U373MG-CIITA cells. Supernatants (from experiment depicted in Fig. 3) from U373MG-CIITA cells infected with adenoviruses expressing GFP-IE1 ({diamondsuit}), GFP-IE1 (86–131) ({blacksquare}), and GFP-IE1M ({blacktriangleup}) together with Ad-Tet-Trans (MOI 20) were used to pulse fresh U373MG-CIITA cells for 3 h. U373MG-CIITA cells infected with Ad-GFP-IE1, Ad-GFP-IE1 (86–131), and Ad-GFP-IE1M but not with Ad-Tet-Trans were used as negative controls (control AdenoV). U373MG-CIITA cells incubated in the presence of IE1 peptide (88–101) (•) or in the absence of peptide ({circ}) were used as positive and negative controls, respectively. U373MG-CIITA cells were then fixed and FzD11 IE1-specific CD4+ T cell clone was added. Activation of CD4+ T cell clone FzD11 in the presence of infected U373MG-CIITA cells was measured in a [3H]thymidine assay (A) and IFN-{gamma} production assay (B).

 
Endogenous presentation of the HLA-DR3 epitope of IE1 is dependent on proteasome and acidic compartments

To determine whether the acidic compartment and proteasomal degradation were involved in the generation of MHC II-restricted peptides, we used inhibitors of these pathways. We used MG132, an inhibitor of the 26S proteasome and chloroquine, which inhibits endosomal processing by blocking acidification of endosomal/lysosomal compartments.

We tested the 24-h p.i. time point when presentation was essentially endogenous because at later time points the presentation process was mixed endoexogenous, thus making difficult to interpret. Data in the absence of inhibitor again confirmed potentiation of the response to GFP-IE1 (86–131) seen in Fig. 3 (data not shown). As shown in Fig. 5A (proliferation), both MG132 and chloroquine impaired presentation of endogenous IE1, and this effect was not affected by IE1 protein localization or size. When used alone, MG132 worked slightly better than chloroquine for the inhibition of responses to GFP-IE1 (86–131) and GFP-IE1M. As controls, FzD11-proliferative responses in the presence of APC pulsed with peptide were not modified in the presence of any of the inhibitors, in agreement with published data showing that chloroquine has no influence on presentation of peptide, whatever its concentration (23). With regard to IFN-{gamma} production (Fig. 5B), inhibitions were more pronounced than that of T cell proliferation. MG132 completely abrogated IFN-{gamma} production in all conditions. The response to peptide was somewhat impaired by MG132 (53% of the response observed in the absence of inhibitor), but this inhibition was not reproduced to the same extent in the presence of MG132 + chloroquine (76% of the response observed in the absence of inhibitor) (Fig. 5B). Thus, the complete abrogation of CD4+ IFN-{gamma} responses to IE1 variants in the presence of MG132 was due to specific inhibition of Ag processing.



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  		FIGURE 5. Presentation of epitope is dependent on proteasome and acidic compartments. U373MG-CIITA cells were incubated with adenoviruses expressing GFP-IE1, GFP- IE1 (86–131), and GFP-IE1M, at various MOI, together with Ad-Tet-Trans (MOI 20) for 3 h. U373MG-CIITA cells were then treated with MG132 (20 µM) and chloroquine (80 µM) during 24 h, fixed, and FzD11 IE1-specific CD4+ T cell clone was added. Activation of CD4+ T cell clone FzD11 in the presence of infected U373MG-CIITA was measured in a [3H]thymidine assay (A) and an IFN-{gamma} production assay (B). ND, not done: values in the absence of inhibitor were too low to evaluate inhibition.

 
Enhanced proteosomal degradation of GFP-IE1 (86–131)

To determine whether the enhanced ability of GFP-IE1 (86–131) to be presented was due to increased degradation/processing, cells expressing different GFP-IE1 isoforms were analyzed by flow cytometry for expression of GFP fluorescence as a marker of the level of protein degradation. As shown in Fig. 6, the basal intensity of staining was higher in cells infected with Ad-GFP-IE1 and Ad-GFP-IE1M than in cells infected with Ad-GFP-IE1 (86–131), suggesting a higher degradation of GFP-IE1 (86–131). Therefore, the CD4+ T cell response was potentiated against the HLA-DR3 epitope despite a lower basal GFP-IE1 (86–131) fluorescence intensity. Moreover, when cells were treated with MG132, the intensity of GFP staining was increased in cells infected with Ad-GFP-IE1 (86–131) but not in cells infected with Ad-GFP-IE1 and Ad-GFP-IE1M. Chloroquine did not cause the same effect of stabilizing GFP protein. Therefore, it appeared that MG132 strongly increased the stability of GFP-IE1 (86–131), suggesting that it was mainly degraded by the proteasome, which may account for the lower basal fluorescence seen with GFP-IE1 (86–131) compared with GFP-IE1 and GFP-IE1M and increased processing and presentation observed in Fig. 3.



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  		FIGURE 6. Enhanced proteosomal degradation of GFP-IE1 (86–131). U373MG-CIITA cells were infected with adenoviruses expressing GFP-IE1, GFP- IE1 (86–131), and GFP-IE1M (MOI 60) together with Ad-Tet-Trans (MOI 20) for 48 h and treated with MG132 (20 µM) and chloroquine (80 µM) for the last 20 h. GFP fluorescence was examined by flow cytometry.

 
Intracellular degradation of GFP-IE1 (86–131) vs GFP-IE1

We next performed pulse-chase/immunoprecipitation experiments to confirm that degradation of GFP-IE1 (86–131) was higher compared with that of GFP-IE1. For this assay, cells expressing GFP-IE1 or GFP-IE1 (86–131) proteins were pulsed for 1 h, then immunoprecipitated using an anti-GFP mAb. As seen in Fig. 7, a rapid degradation of GFP-IE1 (86–131) was observed, as compared with GFP-IE1. By 1 h >40% of GFP-IE1 (86–131) was degraded, and by 5 h there was a >90% reduction in GFP-IE1 (86–131). However, moderate degradation of GFP-IE1 was observed but only at 5 h (45% reduction in protein levels), suggesting that the increased T cell response to GFP-IE1 (86–131), which is located in the cytoplasm, correlates with an increase in degradation.



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  		FIGURE 7. Stability of GFP-IE1 and GFP-IE1 (86–131). U373MG-CIITA cells were infected with adenoviruses expressing GFP-IE1 and GFP-IE1 (86–131) together with Ad-Tet-Trans (MOI 20). Stability of GFP-IE1 and GFP-IE1 (86–131) was evaluated in a pulse-chase experiment. Proteins were immunoprecipitated using an anti-GFP mAb and analyzed by SDS-PAGE. n.i., Not infected; P, pulse.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this article, we have examined the CD4+ T lymphocyte response toward a HLA-DR3-restricted epitope of the HCMV nuclear IE1 protein to better understand the mechanisms of presentation of nuclear proteins and enhance the efficacy of a vaccine directed against HCMV-IE1. We have identified a novel NLS within IE1 exon 4 and have constructed a series of IE1 variants to compare the proliferation of a CD4+ T cell clone in response to the epitope either in its usual nuclear location or routed to the cytoplasm as a shortened or a regular size protein. Based on the use of recombinant adenoviruses and of a model of Ag presentation of an IE1 epitope to a CD4+ T cell clone, we showed that different routage of proteins did not per se modify the intensity of the CD4+ T cell response but did change their capacity to be presented endogenously or exogenously. However, strong potentiation of the CD4+ T cell response was observed when the IE1 epitope was both shortened and routed outside of the nucleus.

By making a panel of IE1 mutations, we have identified a second NLS encoded by exon 4. The newly identified NLS exhibits basic residues classically found in bipartite NLS (24, 25). In the present study, basic residues 326-KR-327 and 340-KRR-342 are separated by 12 residues. Thus, IE1 has evolved to possess at least two NLS, one encoded by exon 2 (18), shared by IE1 and IE2 (6), and the other, bipartite, specific to IE1, i.e., encoded by exon 4 (this study). We have shown previously that IE1 can be endogenously presented to CD4+ T cells and that this interaction leads to the control of infection (15).

When we compared the activation of the IE1-specific T cell clone by APC infected with adenovirus, we demonstrated a higher (10- to >30-fold) response against APC infected with Ad-GFP-IE1 (86–131) compared with APC infected with Ad-GFP-IE1 or Ad-GFP-IE1M. We used an adenovirus expression system, which allowed for control of the level of GFP-IE1 protein expression. The level of protein expression, which was similar in all constructs (data not shown), was thus not responsible for the differences observed in the CD4+ T cell response. In addition, the need for a Trans virus for expression of HCMV proteins by the adenovirus vectors excluded the possibility of the presence of soluble IE1 proteins in the inoculum. The localization of proteins, cytoplasmic vs nuclear, seemed not to be important for the intensity of presentation because there was no difference based on the activation of T cell clone using APC expressing GFP-IE1 (nuclear) and GFP-IE1M (cytoplasmic). However, localization affected the mode, exogenous vs endogenous, of presentation because presentation of GFP-IE1 was more dependent on an endogenous pathway than GFP-IE1M. In contrast, given a cellular localization, the size of the protein was important for its presentation as deduced from the higher response of T cells against APC expressing GFP-IE1 (86–131) compared with APC expressing a larger cytoplasmic protein, GFP-IE1M.

Exogenous Ags, in the form of inactivated virus or soluble protein, can be efficiently processed by APC, such as B cells, macrophages, and dendritic cells, for presentation by class II molecules. However, presentation of endogenous Ags by MHC II molecules has been extensively documented (26). Therefore, our data regarding cytoplasmic GFP-IE1M and GFP-IE1 (86–131) are in accordance with numerous earlier reports reviewed by Aichinger et al. (26). Regarding nuclear GFP-IE1 protein, our present data parallel those obtained with HCMV infection in our group (15). Moreover, they confirm previous observations that a nuclear protein can be endogenously presented (16, 27). Presentation of nuclear IE1 was dependent on both the proteasome and acidic compartments. This is the first demonstration of processing of endogenous nuclear protein by the proteasome. This is at odds with EBNA1 presentation, which is processed through autophagy and degradation by endolysosomes (27), but confirms that endogenously presented proteins can use the proteasome degradation pathway for MHC II presentation (28, 29, 30). It is noteworthy that chloroquine inhibited presentation but did not increase protein expression in contrary to MG132. This may reflect the fact that degradation of the invariant chain, required for Clip peptide release, is dependent on acidic compartments (31). Thus, inhibition of Clip peptide release by chloroquine may prevent antigenic peptide binding to MHC II without influencing IE1 protein stability. Endogenous presentation of IE1 observed in HCMV infection (15), and confirmed here using adenovirus-mediated expression, at least before cytopathic effects lead to release of soluble Ag, is likely to contribute to the focusing of the CD4+ T cell response to the infected cell. As in the case of EBNA1, this may contribute to a better control of infection.

Although processing of epitope from all three proteins studied was dependent on the proteasome, more rapid degradation was established for GFP-IE1 (86–131). Those data are in accordance with reports showing that short cytosolic proteins are degraded more rapidly by the proteasome (25). This may account for higher availability of peptide to be loaded by MHC II proteins. However, it remains to be determined how such generated peptides reach MHC II vesicles for loading. Our data cannot exclude that this is done, as for EBNA1, through autophagy of processed proteins (27).

Vaccination against HCMV is expected to be useful in preventing congenital infections and transplantation-related HCMV diseases. Several strategies have been explored (5). Protection of women of childbearing age would require a vaccine essentially inducing strong Ab production directed toward envelope glycoproteins such as gB (7). Patients with persistent infection may benefit from vaccines eliciting T lymphocyte responses, as proposed by different studies using dense bodies (32), canarypox virus recombinant for pp65 (33), attenuated poxviruses (34), recombinant adeno-associated virus (35), pp65-derived immunodominant peptide (36), and a fusion IE1-pp65 protein (13). Furthermore, in anti-HCMV cellular therapy of bone marrow transplantations such as described by several laboratories (37, 38, 39, 40), it may be of interest to immunize bone marrow donors to induce anti-HCMV T lymphocytes to be infused in the recipient or to increase their frequency. In any case, using recombinant proteins in vaccination would avoid the risk of injecting potentially harmful viral DNA sequences or attenuated laboratory strains that may revert to tropism for endothelial cells, as shown recently (41).

Because IE1 and pp65 contribute to a great part of the anti-HCMV T lymphocyte response (8, 9, 10, 11, 12, 13), it can be argued that a vaccine targeting T lymphocytes should include those Ags. Recent observations indicate that 40% of the total response may be directed toward Ags other than IE1 and pp65 in latently infected individuals (24). Future experiments will determine whether those recently identified epitopes should be also included in a vaccine. Whether the CD8+ T cell compartment can be potentiated in a similar fashion as the CD4+ T cell compartment by deletion of NLS is under investigation. Several strategies have been suggested to increase the CD8+ T cell response: those have used the OmpA protein derived from Klebsiella pneumoniae (42) and Shiga toxin (43). Other examples of increased presentation through increased degradation by ubiquitin (44, 45) have been described and may represent an alternative to increased presentation to and recognition by T lymphocytes. Our data obtained using fully mature, differentiated, CD4+ T cells represent a model of potentiation of T cells. The exploration of the pathway of presentation as well as the capacity to potentiate the initiation phase of naive CD4+ T cells will be useful for the design of future immunization procedures and for the understanding of the response to a nuclear protein. Although the U373MG-CIITA cells we have used as APC are not as potent as dendritic cells, they represent a model, which may serve as a basis for recognition by CD4+ T cells. Increasing the level of priming of CD4+ in vaccines may lead to more efficient protection against infection through both intrinsic antiviral effects of CD4+ T lymphocytes (15, 22) and help toward the CD8+ T lymphocyte response (37, 39).

There is increasing interest upon the CD4+ T lymphocyte response against HCMV: IFN-{gamma} produced by CD4+ T cells participate to the protection against HCMV (46), and there is emergence of granzyme B+CD4+ T cells after recovery of primary HCMV infection (47). Because APC such as dendritic cells and macrophages can be productively infected by HCMV (48), response of CD4+ T cells against Ags produced hours after infection, such as IE1, may be critical for the containment of infection. Moreover, focused interaction of CD4+ T cells toward infected APC, resulting from endogenous presentation, may result in a more efficient control of infection. The transition from endogenous to exogenous presentation, due to cytopathic effects of viral infection may, however, result in a bystander effect of CD4+ T cells toward a broad population of uninfected MHC II-expressing cells and lead to inflammation and pathology. Therefore, it is crucial that the viral infection be controlled before such cytopathic effect occurs. With regard to vaccination, targeting the expression of immunogenic HCMV proteins, such as IE1 and pp65, to professional APC using noncytopathic vectors may also lead to better efficiency of immunization.

In conclusion, we have manipulated the HCMV IE1 protein to increase the CD4+ T cell response toward the HLA-DR3-restricted epitope. This will be of importance in immunization protocols against HCMV infection using for example shorter proteins containing a combination of several specific epitopes. This approach will also be useful to enhance the presentation of other viral nuclear proteins recognized by CD4+ T cells.


   Acknowledgments
 
We thank Denis Hudrisier for critical reading of the manuscript and Christian Davrinche for discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from Institut National de la Santé et de la Recherche Médicale (Action Thématique Concertée), "Région Midi-Pyrénées," "Etablissement français des Greffes," and "Association pour la Recherche sur le Cancer" (ARC). S.D. was supported by the "Ministère de l’Education et de la Recherche" and ARC. Back

2 Current address: Université François Rabelais de Tours, UFR de Médecine, 10 Boulevard Tonnellé, 37032 Tours Cedex, France. Back

3 Address correspondence and reprint requests to Dr. Jean-Luc Davignon, Institut National de la Santé et de la Recherche Médicale Unité 563, Centre de Physiopathologie de Toulouse Purpan, Institut Fédératif de Recherche 30, Centre Hospitalier Universitaire Purpan, 31024 Toulouse Cedex, France. E-mail address: davignon{at}toulouse.inserm.fr Back

4 Abbreviations used in this paper: HCMV, human CMV; MHC II, MHC class II; EBNA1, EBV-encoded nuclear Ag 1; NLS, nuclear localization sequence; EGFP, enhanced GFP; p.i., postinfection. Back

Received for publication June 15, 2005. Accepted for publication September 1, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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