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The Journal of Immunology, 2005, 175: 6481-6488.
Copyright © 2005 by The American Association of Immunologists

Disruption of MHC Class II-Restricted Antigen Presentation by Vaccinia Virus1

Ping Li, Nan Wang, Delu Zhou, Christina S. K. Yee, Cheong-Hee Chang, Randy R. Brutkiewicz and Janice S. Blum2

Department of Microbiology and Immunology, Center for Immunobiology, and Walther Oncology Center, Indiana University School of Medicine, and Walther Cancer Institute, Indianapolis, IN 46202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Vaccinia virus (VV), currently used in humans as a live vaccine for smallpox, can interfere with host immunity via several discrete mechanisms. In this study, the effect of VV on MHC class II-mediated Ag presentation was investigated. Following VV infection, the ability of professional and nonprofessional APC to present Ag and peptides to CD4+ T cells was impaired. Viral inhibition of class II Ag presentation could be detected within 1 h, with diminished T cell responses dependent upon the duration of APC infection and virus titer. Exposure of APC to replication-deficient virus also diminished class II Ag presentation. Virus infection of APC perturbed Ag presentation by newly synthesized and recycling class II molecules, with disruptions in both exogenous and cytoplasmic Ag presentation. Virus-driven expression of an endogenous Ag, failed to restore T cell responsiveness specific for this Ag in the context of MHC class II molecules. Yet, both class II protein steady-state and cell surface expression were not altered by VV. Biochemical and functional analysis revealed that VV infection directly interfered with ligand binding to class II molecules. Together, these observations suggest that disruption of MHC class II-mediated Ag presentation may be one of multiple strategies VV has evolved to escape host immune surveillance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Major histocompatibility complex class II complexes composed of {alpha}{beta} heterodimers function to bind and display peptides derived from exogenous or endogenous Ag for recognition by CD4+ T lymphocytes. Such presentation is central to initiate primary, cellular immune responses as well as to establish efficient, durable secondary immune responses (1, 2). Although long known to mediate protective immunity to bacteria and toxins, the important roles of CD4+ T cells in tumor immunity and viral clearance have also been well established (3, 4, 5, 6, 7).

Vaccinia virus (VV),3 a member of the poxvirus family with considerable homology to smallpox and cowpox, has been used as an attenuated vaccine to successfully eradicate human smallpox. With DNA replication exclusively in the cytosol and the ability to induce both cellular and humoral immunity, VV has also been championed as a potential live recombinant vaccine vector to promote immunity against tumors and other infectious disease (8). However, VV has been shown to also interfere with the host protective immunity (9). VV blocks cytokine signaling by expressing secreted decoy receptors for IL-1{beta}, TNF-{alpha}, IFN-{alpha}{beta}, and IFN-{gamma} (10, 11). VV protein A52R targets TLR signaling complexes to suppress host defense (12). The vaccinia virus complement-control protein can inhibit both the classical and alternative pathways of complement activation for the purpose of evading the host immune responses (13). A very early, transient decrease in T cell responses in healthy humans has been reported after VV exposure (14). Immunosuppression was also observed when recombinant VV (rVV) was used as a therapeutic vaccine for head and neck cancer in a murine model (15). These and other observations suggest that VV infection might induce defects in APC. It has been previously shown that VV infection can perturb MHC class I-mediated Ag presentation (16, 17, 18, 19). This inhibitory effect is selective for certain epitopes and can be overcome by enhancing Ag degradation, pointing to alterations in Ag processing (17). Studies also indicate that VV-infected APC fail to present certain class II-restricted epitopes (20, 21, 22). Yet the specific means by which viral infection perturbs APC function remain poorly defined. Dissecting the mechanisms by which VV subvert cellular immune response is essential for the production of safer and more immunogenic vaccines.

In this study, evidence is provided that VV can disrupt MHC class II-mediated Ag presentation. A human HLA-DR4+ B lymphoblastoid cell line (B-LCL) rapidly lost its ability to present an endogenous cytoplasmic autoantigen glutamate decarboxylase (GAD) to CD4+ T cells after VV infection. Encoding this Ag within the virus behind an early/late promoter ensured rapid and sustained production of GAD in infected APC, yet failed to promote T cell responsiveness. Viral inhibition of Ag presentation was dependent on the duration of infection and virus titer, yet could be observed with replication-deficient virus. Provision of external costimulation could not restore T cell responses to infected APC. VV infection of APC perturbed the presentation of exogenous peptides as well as Ag in the context of human or murine class II alleles. Western blot analysis revealed that VV infection did not alter steady-state levels of endogenous Ag or class II proteins within APC. Flow cytometry further confirmed no change in surface class II levels after VV infection of APC. Biochemical and functional assays indicated that VV infection perturbed peptide binding to cell surface class II molecules. Thus, disruption of peptide loading via MHC class II molecules may be one of several strategies VV has evolved to disrupt APC function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Virus and cells

VV Western Reserve strain, TK–143B (a human osteosarcoma cell line), and CV-1 (African green monkey kidney cell) were provided by Drs. J. Yewdell and J. Bennink (National Institutes of Health, Bethesda, MD). rVV was generated in TK–143B cells. VV was propagated and titered using CV-1 cells. Both cells were cultured in DMEM (Invitrogen Life Technologies) supplemented with 10% FBS, 50 U/ml penicillin, 50 µg/ml streptomycin, and 2 mM L-glutamine. VV was released from infected cells by three cycles of freezing/thawing, sonication, and clarification by centrifugation. This VV preparation was further purified on a 36% sucrose gradient by ultracentrifugation. Lysates from uninfected CV-1 cells were similarly prepared for use as a control for mock infection. In some experiments, purified virus was treated with UV light (254 nm for 20 min). Studies confirmed UV-treated virus could infect cells but failed to replicate. The B-LCL Priess (homozygous for HLA-DR4), PriessGAD (retrovirally transduced for constitutive expression of the 65-kDa form of human GAD), and THP-1.DR4 (human monocytic cells, retrovirally transducted with DR4) were maintained in IMDM (Invitrogen Life Technologies) supplemented with 10% heat-inactivated calf serum. LDR4 (fibroblast L cell, retrovirally transducted with DR4) was maintained in DMEM supplemented as above. Bone marrow-derived dendritic cells (DC) were prepared as previously described (23). Briefly, total bone marrow cells were extracted from mouse femurs and tibiae and depleted of erythrocytes, T cells, B cells, and other MHC class II-positive cells. Bone marrow precursors were then cultured for 5 days in RPMI 1640 supplemented with 5% FBS, 10 ng/ml recombinant mouse GM-CSF (BD Pharmingen), and 20 µg/ml gentamicin. After 48 h of culture, nonadherent cells were removed carefully and the loosely adherent immature DC (IMDC) were tested for virus infection and Ag presentation. The mouse B cell line 1153 (I-Ab) and T cell hybridomas, including 33.1 specific for GAD273–285 and HLA-DR4, 17.9 specific for HSA64–72 and HLA-DR4, and B04 specific for HEL74–88 and I-Ab, were maintained in RPMI 1640 (Invitrogen Life Technologies) supplemented as above.

Peptides

GAD273–285 (IAFTSEHSHFSLK), GAD273–285-biotin (GAD-B), HEL74–88 (NLCNIPCSALLSSDI), HSA64–76 (VKLVNEVTEFAKTK), and {kappa} I188–203 (KHKVYACEVTHQGLSS) were produced using F-moc technology and HPLC to ensure purity of >90% (24, 25).

Generation of rVV-expressing GAD protein

The human autoantigen GAD was expressed in rVV under the control of the early/late promoter P7.5 to ensure expression in virally infected APC. Since there is a VV early transcription termination signal TTTTTNT in the middle of GAD cDNA, a point mutation was introduced using a QuikChange Site-Directed Mutagenesis kit (Stratagene) according to the manufacturer’s protocol. Two mutagenic primers (with nucleotide changes underlined) were synthesized: 5'-GAA CAT AGT CAT TTC TCTCTC AAG AAG GG-3' (sense) and 5'-CCC TTC TTG AGA GAG AAATGA CTA TGT TC-3' (antisense). Silent mutation of GAD was confirmed by DNA sequencing. Mutated GAD cDNA was excised from pBluescriptGAD by NotI and inserted into the NotI site of pSC11. Plasmid orientation of GAD under the control of vaccinia P7.5 promoter was confirmed by DNA sequencing. Recombinant virus was generated as described elsewhere (26). Briefly, VV (Western Reserve)-infected TK–143B cells were transfected with plasmid pSC11GAD containing the mutant GAD flanked by the VV TK sequence to provide sites for homologous recombination. rVV (rVVGAD) was characterized by Western blot using a specific polyclonal GAD Ab (Sigma-Aldrich).

Ag presentation assays

APC were infected with VV at a multiplicity of infection (MOI) of 5 for 6 or 24 h. No cytopathic effect was observed with APC at 6 h and very limited loss of cell viability was found at 24 h. To study exogenous class II presentation, VV- or mock-infected APC were incubated with increasing concentrations of peptides or Ag at various time points, followed by washing, fixation (0.5% paraformaldehyde, 10 min, 4°C), and cultivation with specific T cells. To investigate the effect of VV on peptide binding to surface class II molecules, Priess cells were preloaded with GAD peptide overnight and infected with VV (MOI = 5) for 6 h. To monitor the effect of viral infection on endogenous Ag processing and presentation, APC were VV- or mock-infected at different times, fixed, and cocultured with T cells for 24 h. As a control, all studies were conducted using mock-infected APC or APC incubated in fresh medium. For mock treatments, APC were cultured in medium supplemented with a fractionated extract from CV-1 cells. No difference in Ag presentation was observed upon APC exposure to varying amounts of this fractionated CV-1 cell lysate. In some studies, to provide external costimulation, T cells were pretreated with anti-CD28 mAb to cross-link CD28 before incubation with virally infected APC. To test whether VV-infected APC could affect T cell activation of virus-free APC function, PriessGAD were mixed with fixed-infected or uninfected Priess cells before coculture with specific T cells. To investigate the effect of VV infection on preexisting peptide-class II complexes, Priess cells were pulsed with increasing concentrations of GAD273–285 peptide overnight, then infected with VV (MOI = 5) for 6 h. Infected Priess were cocultured with specific T cells. T cell IL-2 production was quantitated using HT-2, an IL-2-dependent T cell line as previously described (27). HT-2 cell proliferation was quantitated via [3H]thymidine incorporation. Data are expressed as cpm with all assays performed in triplicate and the mean and SD calculated. Each value is representative of a minimum of three independent experiments.

Electrophoretic analysis and immunoblotting

Cells infected with VV (MOI = 5) or treated under mock conditions for different times were harvested for Western blot analysis. An electrophoretic assay was designed to measure peptide binding to class II molecules. Briefly, VV- or mock-infected APC were incubated with 10 µM GAD-B for 6 h. These cells were lysed in SDS sample buffer with or without boiling. Biotin-peptide (GAD-B) binding to class II dimers was detected via SDS-PAGE. The identity of these complexes was confirmed by immunoprecipitation with the DR-specific mAb L243 (28). For the peptide-binding assay and analysis of class II and Ag expression in VV- or mock-infected APC, samples of 80–100 µg of total cell protein were fractioned by SDS-PAGE followed by transfer to nitrocellulose membranes. Samples were probed for GAD expression using a rabbit anti-human GAD polyclonal Ab (Sigma-Aldrich) or DR{alpha}{beta} dimers using the mAb L243. GAD, DR{alpha}{beta} dimers, or GAD-B peptide were visualized using the secondary reagents goat anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP (Jackson ImmunoResearch Laboratory) or streptavidin-HRP (Pierce) followed by ECL (Amersham Pharmacia Biotech). Cellular actin and GAPDH expression were examined as controls using Pan Actin Ab-5 (Lab Vision) and anti-GAPDH Ab from Chemicon International as described above.

RNA isolation and RT-PCR analysis

PriessGAD infected with purified VV (MOI = 5) for 0, 2, and 6 h were harvested, and total RNA was isolated using an RNEasy Mini kit (Qiagen) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 0.6 µg of total cellular amounts of RNA using an Advantage RT for PCR kit (BD Biosciences Clontech). For PCR amplification, primers were designed for E3L as follows: 5'-ACG AGC GTT CTA ACG CAG AG-3' (sense) and 5'-AAT GAT GAC GTA ACC AAG AAG TTT-3' (antisense). GAPDH primers were used as a control, provided by an Advantage RT for PCR kit (BD Biosciences Clontech). Amplification reactions were set up in Platinum PCR SuperMix (Invitrogen Life Technologies) and performed in a MJ Research thermal cycler (model PTC-100TM) using the following parameters: 94°C, 45 s; 60°C, 45 s; 72°C, 45 s, 28 cycles. Samples (10 µl) were separated on a 1.6% agarose, and amplified products were detected by ethidium bromide staining and UV transillumination.

Fractionation of VV

VV isolated directly from CV-1 cells was loaded into the sample reservoir of Microcon-10 filter chambers with a 10-kDa cutoff membrane (Amicon) and centrifuged at 10,000 x g with collection of low-molecular mass filtrate and high-molecular mass retentate. Equal volumes of each fraction were added to Priess cells, and T cell assays were performed. CV-1 cell lysate and unfractionated VV were used as controls.

Flow cytometry

For surface HLA-DR staining, cells were incubated with L243 or an isotype-matched irrelevant mAb followed by FITC-conjugated AffiniPure F(ab')2 goat anti-mouse IgG Fc{gamma} fragment-specific Ab (Jackson ImmunoResearch Laboratories). Similar analyses were conducted using the anti-class II mAb HB10.A, two mAbs specific for empty human class II complexes (a gift from L. Stern, University of Massachusetts Medical School, Worcester, MA), and anti-mouse I-Ab mAb Y3P. After staining, cells were fixed in 0.5% paraformaldehyde. During FACS, samples were gated for analysis of single cells using a FACScan with CellQuest software (BD Biosciences). To monitor viral protein expression in infected APC, cells were infected with VV (MOI = 5) for 6 h and stained for D8L (polyclonal Ab provided by Dr. W. Chang, Taiwan) or E3L (mAb Tw2.3 provided by Drs. J. Yewdell and J. Bennink, National Institutes of Health), followed by PE-conjugated AffiniPure F(ab')2 goat anti-rabbit IgG Ab or FITC-conjugated AffiniPure F(ab')2 goat anti-mouse IgG Fc{gamma} fragment-specific Ab (Jackson ImmunoResearch Laboratories).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
VV infection of human B cells disrupts class II-restricted cytoplasmic Ag presentation to CD4+ T cells

Viral evasion of host immune responses via disruption of MHC class I Ag presentation is well established (29). Recent studies have demonstrated alterations in class II Ag presentation as a result of viral infection (30, 31, 32, 33). To investigate the effect of VV on class II-restricted Ag presentation and T cell activation, a DR4+ B-LCL, PriessGAD producing an endogenous autoantigen GAD was used as a model APC. GAD is localized in the cytoplasm of these APC and undergoes processing by the proteasome and calpain (24). GAD epitopes are then translocated into endosomes for binding to class II molecules before surface expression and presentation to T cells (34). Following VV infection of PriessGAD, a progressive reduction in GAD presentation was observed over the time (Fig. 1A). Vaccinia early gene E3L mRNA could be detected after a 2-h infection of APC by RT-PCR (Fig. 1B). However, Western blot and densitometric analysis revealed that the steady-state levels of GAD in APC were not altered for at least 6 h after VV infection (MOI = 5). At this same time point, the presentation of GAD was decreased by virus ~60% (Fig. 1A). Inhibition of Ag presentation was found as early as 1 h after PriessGAD cells were exposed to VV (data not shown) and was more extensive during late phases of infection (i.e., >2 h). To exclude the possibility that CV-1 cells used to propagate the virus contribute to this inhibitory effect, APC were exposed to a fractionated cell lysate prepared from mock-infected CV-1 cells, and no reduction in GAD presentation was observed compared with cells exposed to fresh medium (Fig. 1B). To determine whether the inhibition of class II presentation was linked to a virus-induced disruption of GAD protein synthesis, a rVVGAD encoding GAD cDNA under the control of a viral promoter was generated and used to infect the B-LCL, Priess. GAD expression in APC increased during the course of rVVGAD infection (Fig. 1C). But these infected APC failed to present the VV-derived GAD to T cells despite measurable production of newly synthesized Ag (Fig. 1D). To test whether the inhibition was dependent on viral replication, VV was treated with UV light (254 nm). UV-treated VV retained its ability to disrupt endogenous Ag presentation (Fig. 1E). This result suggests that the inhibitory factor is likely carried into APC with assembled virions. The observation that live virus is more effective in inhibiting class II Ag presentation may indicate virus replication increases the inhibitory factor in infected APC. Inhibition of class II autoantigen presentation by VV was dependent on the dose of virus used to infect APC, although at high viral MOI this inhibitory effect plateaued. This may reflect saturation of virus-host cell interaction at higher MOI (Fig. 1E). Viral inhibition was also observed using varying numbers of infected APC in T cell assays (Fig. 1F).



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  		FIGURE 1. VV infection of APC blocks T cell recognition of a cytoplasmic Ag in the context of MHC class II molecules. A, Endogenous GAD presentation in VV-infected APC. PriessGAD, a B-LCL expressing the cytoplasmic Ag GAD, was infected with purified VV (MOI = 5) for 2–24 h. PriessGAD were cultured in fresh medium and used as a control (no VV). All APC were treated with paraformaldehyde before incubation with T cells. Complexes of antigenic peptides and class II DR4 on the surface of APC were quantitated by monitoring T cell hybridoma activation and IL-2 production. APC expression of GAD Ag and endogenous actin were monitored by Western blot (insert). No significant reduction in the ratio of GAD Ag expression relative to host cell actin levels was observed as determined by densitometry in three replicate experiments. B, Comparison of endogenous GAD presentation using APC in fresh medium, treated with fractionated CV-1 cell lysate, or VV. PriessGAD were cultured in fresh medium, medium supplemented with a partially purified extract from CV-1 cells, or VV (MOI = 5) for 6 h, then fixed in 0.5% paraformaldehyde, and cocultured with GAD-specific T cells. T cell proliferation was measured as described. Transcription of viral early gene E3L or GAPDH in VV-infected APC was analyzed by RT-PCR (insert). Total RNA was isolated at the indicated times. C, Time course of GAD protein synthesis upon rVVGAD infection of APC. Priess were infected with rVVGAD (MOI = 5) for 2–12 h. Samples (80 µg) of total cell protein were resolved on 10% SDS-PAGE followed by immunoblotting with a GAD-specific Ab. Uninfected Priess (time 0) and retrovirally transduced PriessGAD were used as negative and positive controls, respectively. D, APC infected by rVV expressing GAD Ag (rVVGAD) failed to activate T cells. Priess were infected with rVVGAD (MOI = 5) for 6 h, fixed, and cocultured with specific T cells. Priess (control) or GAD peptide-pulsed Priess cells were also tested for T cell activation. E, UV-inactivated VV inhibited endogenous GAD presentation. Purified VV was treated with UV light (254 nm) for 20 min on ice. PriessGAD were infected with live- or UV-inactivated VV (MOI = 1, 5, 10) for 6 h. Cells were fixed and cocultured with specific T cells. GAD presentation was measured as described. Infection of APC at varying doses of live VV was confirmed by monitoring the expression of VV proteins (35 kDa, H3L) detected with a polyclonal anti-virus Ab by Western blot (insert). F, Assays with variable APC:T cell ratios showed a similar inhibition pattern for GAD presentation. PriessGAD were infected with purified VV (MOI = 5) for 6 h, then fixed in 0.5% paraformaldehyde, and cocultured with GAD-specific T cells at the indicated ratios. Data from T cell assays are representative of results from three to five individual experiments with SD from the analysis of triplicate samples within each experiment. Open columns represent uninfected APC, and filled columns are results from virus-infected APC.

 
VV-induced APC dysfunction is not linked to diminished costimulation or transfer of a negative signal from APC to T cells

To test whether VV infection affects the costimulation signal by APC, T cells were pretreated with an anti-CD28 mAb to cross-link CD28 before incubation with mock- or VV-infected PriessGAD. Previous studies have shown such an external signal can compensate when APC expression of costimulatory molecules is low (35, 36). Provision of this external costimulation failed to rescue Ag presentation in VV-infected APC (Fig. 2A). Studies were also conducted to test whether viral infection of APC induces host or viral factors which can deliver a negative signal or unresponsiveness in T cells. Yet, T cell responses were not disrupted when APC-expressing Ag were mixed with aldehyde-fixed infected APC (Fig. 2B). To determine whether low-molecular mass factors secreted or associated with the virus are responsible for inhibition of Ag presentation, virus preparations were fractionated into low-molecular mass VV (LMVV <10 kDa) and high-molecular mass fraction of VV (HMVV) components. The volume of each of these fractions was restored to that of the original virus preparation before filtration. APC were incubated with these fractions (6 h) and decreased T cell responses were observed only when APC were exposed to the HMVV (Fig. 2C). These results suggest that viral components (>10 kDa) or the intact virion are responsible for the disruption of class II function.



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  		FIGURE 2. Interaction of T cells with VV-infected APC. A, Provision of external costimulation signal. T cells were pretreated with anti-CD28 mAb to cross-link CD28 before incubation with mock- or VV-infected PriessGAD (PG). Untreated T cells were cocultured with mock- or VV-infected PriessGAD as a control. B, VV-infected APC did not interfere with T cell recognition of virus-free APC. T cells were cocultured with the following APC: PriessGAD alone, PriessGAD plus Priess, PriessGAD with VV-infected Priess, PriessGAD infected with VV, or Priess treated with VV alone. Only PriessGAD expresses the GAD Ag, and all APC were aldehyde fixed before coculture to prevent virus transmission. C, Fractionation of virus to test for inhibitory function. Virus was passed through a Microcon-10 concentrator (10-kDa cutoff). Fractionated material was collected and equal volumes of the LMVV, and the HMVV were added to APC. APC function was measured by T cell assay. APC incubated with CV-1 cell lysate preparation or VV were the controls in this experiment. Results are representative of three independent experiments with the SD for triplicated samples indicated.

 
Decreased MHC class II-mediated Ag presentation in VV-infected professional and nonprofessional APC

The effect of virus on exogenous peptide presentation by class II molecules was tested using professional APC (IMDC-bone marrow-derived immature DC, macrophages, and B cells) as well as nonprofessional APC (fibroblasts). Typically, exogenous peptides bind directly to available cell surface class II proteins yielding complexes for T cell recognition. Yet some peptides, like whole Ag, require internalization and processing before functional presentation (25, 36, 37). HEL74–88 and {kappa} I188–203, containing cysteine residues, require intracellular processing for reduction of cysteine before functionally binding newly synthesized class II molecules within mature endosomes (36, 37). Both IMDC and macrophages (THP-1) showed defects in presenting HEL74–88 and {kappa} I188–203 peptides after VV infection (Fig. 3, A and B). Macrophages in particular appear to be highly susceptible to viral disruption of class II presentation, even at high doses of antigenic peptides. Macrophages were pretreated with IFN-{gamma} in these experiments before virus infection. Even with the GAD273–285 peptide, which directly binds DR4 molecules on the cell surface (24), virally infected human B cell, fibroblast, and macrophage (DR4+) presentation to T cells was reduced compared with uninfected APC (Fig. 3, C and D; macrophage data not shown). To test whether the inhibition was linked to the efficiency of infection, Abs specific for viral proteins E3L and D8L were used to monitor the percent infected cells by FACS. Professional APC (IMDC, 60%; macrophages, 76%; B cells, 95%) displayed a higher percentage of infected cells potentially accounting for more severe loss of function compared with nonprofessional APC (fibroblasts, 45%). We also investigated the effect of VV on processing and presentation of HSA peptide and Ag by human B cells. HSA64–76, a human serum albumin-derived peptide, must be internalized into an early endosomal compartment where it is processed before binding mature, recycling class II molecules before T cell recognition (25). HSA Ag needs to be internalized and processed in endosomal-lysosomal compartments to yield peptides capable of binding and presentation via class II molecules. Again, a block in HSA64–76 as well as HSA Ag presentation was observed using virally infected APC (Fig. 4). The severity of this inhibition was dependent on both the length of infection and the dose of Ag tested. These results together suggest VV perturbs both exogenous and endogenous routes of class II Ag presentation by professional and nonprofessional APC.



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  		FIGURE 3. VV infection alters T cell responses to peptides presented via MHC class II molecules on professional and nonprofessional APC. Cells were infected with or without VV (MOI = 5) for 90 min, followed by washing and further cultivation with fresh medium as follows: IMDC incubated with HEL74–88 peptide (A); {gamma}-IFN-pretreated macrophage THP-1.DR4 incubated with {kappa} I188–203 peptide (B); B-LCL incubated with GAD273–285 peptide (C); and LDR4 fibroblasts incubated with GAD273–285 peptide (D). After 6 h, APC were fixed in 0.5% paraformaldehyde and cocultured with Ag-specific T cells. T cell activation was assessed by monitoring IL-2 production using HT-2 cells. Results are representative of three independent experiments. Cells were also analyzed by FACS for viral gene products (E3L, D8L) to confirmed percent infection (insert).

 


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  		FIGURE 4. Presentation of epitopes by recycling and newly synthesized class II molecules was disrupted by VV infection. B-LCL were infected with or without VV (MOI = 5) for 90 min, followed by washing and incubation with HSA64–76 peptide or HSA Ag for 6 or 16 h. After incubation, APC were fixed in 0.5% paraformaldehyde and cocultured with Ag-specific T cells for analysis of T cell activation. Results are representative of three independent experiments.

 
VV alters the association of peptides with class II molecules

MHC molecules themselves are targets for virus evasion strategies. Human CMV decreases cell surface MHC class I and class II expression (38, 39). To explore whether a similar mechanism was responsible for APC dysfunction after VV infection, steady-state levels of class II protein expression were determined for virus-infected B-LCL. Western blot analysis revealed no change in steady-state levels of class II dimers at either 6 h or 24 h after VV infection compared with mock-infected controls (Fig. 5A). Surface HLA-DR expression was examined by flow cytometry using B-LCL that were VV or mock infected. VV infection did not affect surface class II DR expression on B-LCL at 6 h (Fig. 5B). Experiments using mAbs recognizing empty class II complexes also failed to reveal any change with viral infection of APC (data not shown). Similarly, class II I-Ab expression on murine B cells was unchanged after viral infection (data not shown). Yet functional assays indicated a significant reduction in T cell recognition of peptide-class II complexes at this time (Fig. 1A). To determine whether VV interfered with peptide binding to surface class II, the association of GAD-B peptide with HLA-DR4 was directly analyzed by electrophoresis. GAD-B was detected as a complex in B cells via SDS-PAGE at a relative molecular mass of 60 kDa as expected. The identity of this peptide-class II complex was previously established by coimmunoprecipitation of GAD-B with HLA-DR (34). Densitometry results demonstrated that GAD-B peptide binding to HLA-DR4 was decreased by 57% in VV-infected Priess cells compared with mock-infected cells using this electrophoretic assay (Fig. 5C). Again, no change was observed in total DR{alpha}{beta} complexes as detected on SDS-PAGE. Functional assays further revealed the expression of preformed GAD peptide-class II complexes could be dramatically disrupted following viral infection of APC (Fig. 5D). Together, these results indicated that perturbation of class II-peptide complexes display and recognition by T cells represents another means by which this DNA virus disrupts host immune responses.



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  		FIGURE 5. VV infection of APC interfered with class II-peptide binding but did not alter class II protein expression. A, Western blot analysis of DR{alpha}{beta} dimers from VV- or mock-infected B-LCL. Priess cells were infected with or without VV (MOI = 5) for 6 or 24 h. HLA-DR{alpha}{beta} dimer expression was detected by mAb L243. The steady-state level of cellular class II molecules was not decreased at 6- or 24-h after viral infection. GAPDH in the same samples was analyzed as a control. B, Flow cytometric analysis of cell surface class II expression in control or virus-infected APC. Priess cells were infected with VV (dotted line) or mock-infected (dark line) as above and stained for surface DR expression with L243 mAb. The shaded histogram represents isotype-matched irrelevant Ab staining of control or infected cells. Surface class II DR expression was unchanged after VV infection. C, Electrophoretic analysis of peptide binding to class II molecules on mock- or VV-infected B-LCL. Mock- or VV-infected Priess cells were pulsed with 10 µM GAD-B for 6 h. Samples were resolved on 10% SDS-PAGE. GAD-B was detected by streptavidin-HRP and ECL. GAPDH and total class II DR{alpha}{beta} in samples were analyzed as a control. Representative blots from three independent experiments with SD from these assays are indicated. D, Analysis of functional preexisting peptide-class II complexes in APC after VV infection. Priess were preincubated with GAD peptide at the indicated concentrations overnight. Cells were washed and infected with purified VV (MOI = 5) for 6 h, then fixed and cocultured with T cells. APC function was measured as described, and results are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Ag processing and presentation by MHC class II molecules are essential for the activation of helper T cells during immune responses. Recent studies indicate that viruses target MHC class II-restricted Ag processing and presentation pathways to render APC ineffective in stimulating T cells and ultimately lead to immune evasion. Published studies have well established the role of MHC class I molecules in activating antiviral immunity as well as the evolution of numerous viral strategies to thwart the class I pathway (29, 40). Yet, the importance of MHC class II molecules in protective immunity against live viruses is also documented (6, 7, 41). Thus, an increasing number of studies also point to viral disruption of MHC class II Ag presentation. Human CMV US2 targets HLA-DR{alpha} and HLA-DM{alpha} for degradation by the proteasome, thus inhibiting Ag presentation to CD4+ T cells (30). HIV Nef impairs MHC class II Ag presentation by increasing immature MHC II and down-modulating mature cell surface MHC II (31). Human papillomavirus 16 E5 protein perturbs MHC class II maturation by inhibiting the degradation of invariant chain (32). EBV gp42 impairs T cell activation by binding to HLA class II molecules and blocking TCR engagement (33). Lymphocytic choriomeningitis virus interferes with DC maturation and Ag-presenting activity by reducing the surface expression of MHC class I, MHC class II, CD40, CD80, and CD86 molecules (42).

In this study, we demonstrated that human B cells lost their ability to present peptides as well as endogenous and exogenous Ag after exposure to VV. Similar inhibition was observed upon VV infection of other professional APC including DC and macrophages as well as fibroblasts, a nonprofessional APC. VV disrupts DC maturation, thus class II peptide presentation by immature DC was tested with or without virus treatment. VV can also block IFN activation of macrophages via disruption of STAT1 function (43). Monocytes were therefore activated first and then exposed to VV to assess changes in class II peptide presentation. In each case, VV infection of DC or macrophages resulted in diminished peptide presentation. VV infection in vitro of B cells, macrophages, and immature DC was efficient, with comparable high percentages of cells expressing viral early and late gene mRNA and proteins. Injection of an enhanced GFP-expressing virus in mice resulted in immunofluorescent detection of VV associated with lymph node macrophages and DC, but not B cells (44). Whether distinct APC are infected by virus delivered during immunization via skin puncture or lung inhalation is not clear. Nonetheless, virus disruption of class II presentation was observed using a panel of Ag and peptides and both human and murine APC, independent of MHC haplotype.

VV may act to thwart several independent steps in class II Ag processing and presentation or disrupt a common essential step shared among distinct pathways for Ag uptake and presentation. CD4 T cell responses to short peptides, native endogenous and exogenous Ag in the context of virally infected APC, were diminished. Exposure of APC to virus also reduced epitope presentation in the context of newly synthesized and recycling class II molecules. Encoding Ag under the control of a VV early/late gene promoter to ensure high levels of expression during infection also failed to overcome the inhibitory effect of the virus. These results suggest VV may disrupt presentation at a stage conserved among each of these distinct class II pathways. UV-inactivated VV, which is unable to replicate, blocked the ability of APC to activate T cells, indicating that disruption of APC function is not dependent on viral DNA replication. This latter finding is consistent with the rapid loss of APC function upon exposure to virus. To determine whether the virus perturbs the interaction of APC and T cells but not MHC recognition, several approaches were taken. In DC, VV can selectively reduce the surface expression of the costimulatory molecule CD80 and the DC maturation marker CD83, consistent with a block in DC maturation (45, 46). Although human B-LCL express constitutive, high levels of costimulatory molecules, external costimulation (cross-linking CD28) was provided to T cells in an attempt to restore class II presentation. However, anti-CD28-treated T cells still showed reduced responses to Ag in the context of infected APC. Thus, APC dysfunction was unlikely due to a blockage in costimulation by VV infection. Coincubation of virally infected APC with uninfected cells containing Ag failed to alter T cell recognition of the latter target cells. Cells were fixed to prevent viral transmission in these studies, yet the experiments suggest VV infection does not induce the expression of a factor on APC which acts directly to thwart T cell activation independent of MHC.

The strength of T cell responses is dependent on the quantity of functional peptide-class II complexes displayed on the surface of APC. Although the overall levels of surface and intracellular class II molecules appeared similar in APC with or without virus, VV-infected APC displayed a reduced capability for class II ligand loading and presentation. Biochemical studies revealed diminished binding of a labeled peptide to class II at the cell surface. Remarkably, VV infection also disrupted the stability of existing peptide-class II complexes on the surface of APC reminiscent of the function of HLA-DM. These studies, in conjunction with functional assays to monitor Ag presentation, suggest that virus may act directly to disrupt peptide-MHC complexes. Whether this is linked with the stability of the class II-peptide complex remains unclear. Viral infection did not diminish the percentage of APC-expressing CLIP-DR complexes, although the level of CLIP-DR complexes displayed as measured by mean fluorescence intensity was slightly decreased in infected cells (data not shown). Diminished class II presentation of short synthetic GAD peptides was observed using live or fixed APC that had been VV infected. Thus, the possibility that viral infection triggers peptide destruction or proteolysis appears less likely. In line with this, the inhibitory effects of VV were observed with a panel of structurally diverse Ag and peptides. Studies are underway to determine whether viral components associate with class II molecules to disrupt ligand binding. Viral peptides generated during infection of APC should also gain access to class II molecules and could compete with exogenous and endogenous Ag, particularly at later stages of viral infection. However, the quick onset of viral inhibition as well as the observation of inhibition with nonreplicating virus would require that abundant, high-affinity viral peptides rapidly displace class II ligands to account for the observed changes in class II presentation using VV-infected APC. To determine whether loosely associated peptides or inhibitory factors were associated with infectious virions, low- and high-molecular mass fractions from VV were separated and tested in functional assays. Yet, only the high-molecular mass fractions containing infectious virus proved inhibitory. Using a predictive algorithm for class II DR4 ligand binding, synthetic peptides from several immunogenic VV Ag were generated and tested for their ability to disrupt GAD peptide binding and display to T cells. Yet, even at >10-fold excess, none of these viral epitopes could disrupt GAD peptide presentation (data not shown). Mass spectral analysis of class II ligands from APC at early and late stages of viral infection should prove informative in this respect. Taken together, studies here indicate direct perturbation of peptide binding to class II may be one of the mechanisms that VV has evolved to disrupt Ag presentation.


   Acknowledgments
 
We thank Dr. V. Crotzer and J. Gregg for critical reading and helpful discussion, Katherine Toomey for technical assistance.


   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 National Institutes of Health (NIH) Grant AI49589, the Indiana Genomics Initiative at IUSM (to J.S.B), NIH Grant AI0560972 (to J.S.B., C.H.C., and R.R.B.), and NIH Grant AI46455 (to R.R.B.). Back

2 Address correspondence and reprint requests to Dr. Janice S. Blum, Department of Microbiology and Immunology, Indiana Medical Science Building, Room 420, University School of Medicine, 635 Barnhill Drive, Indianapolis. E-mail address: jblum{at}iupui.edu Back

3 Abbreviations used in this paper: VV, vaccinia virus; GAD, glutamate decarboxylase; HEL, hen egg lysozyme; LMVV, low-molecular mass fraction of VV; HMVV, high-molecular mass fraction of VV; HPV, human papillomavirus; HSA, human serum albumin; MOI, multiplicity of infection; B-LCL, B lymphoblastoid cell line; DC, dendritic cell; IMDC, immature DC; rVV, recombinant VV. Back

Received for publication May 12, 2005. Accepted for publication August 30, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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