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The Journal of Immunology, 2007, 178, 7557 -7562
Copyright © 2007 by The American Association of Immunologists, Inc.
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Rates of Processing Determine the Immunogenicity of Immunoproteasome-Generated Epitopes1

Parampal Deol2,*, Dietmar M. W. Zaiss2,*, John J. Monaco{dagger} and Alice J. A. M. Sijts3,{dagger}

* D. Smith Center for Vaccine Biology and Immunology and Department of Microbiology and Immunology, University of Rochester, Rochester, NY 14642; and {dagger} Department of Molecular Genetics, University of Cincinnati, Cincinnati, OH 45267


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD8 T cells resolve intracellular pathogens by responding to pathogen-derived peptides that are presented on the cell surface by MHC class I molecules. Although most pathogens encode a large variety of antigenic peptides, protective CD8 T cell responses target usually only a few of these. To determine the mechanism by which the IFN-{gamma}-inducible proteasome (immuno) subunits enhance the ability of specific pathogen-derived peptides to elicit CD8 T cell responses, we generated a recombinant Listeria monocytogenes strain (rLM-E1) that secretes a model Ag encompassing the immunoproteasome-dependent E1B192–200 and immunoproteasome-independent E1A234–243 epitope. Analyses of Ag presentation showed that infected gene-deficient professional APCs, lacking the immunosubunits LMP7/ibeta5 and MECL-1/ibeta2, processed and presented the rLM-E1-derived E1B192–200 epitope but with delayed kinetics. E1A epitope processing proceeded normally in these cells. Accordingly, infected gene-deficient mice failed to respond to the otherwise immunodominant E1B192–200 epitope but mounted normal CD8 T cell responses to E1A234–243 which was processed by the same professional APCs, from the same rLM-E1 Ag. The inability of gene-deficient mice to respond to E1B192–200 was not explained by insufficient quantities of antigenic peptide, as splenic APC of 36-h-infected gene-deficient mice that presented the two E1 epitopes at steady state levels elicited responses to both E1B192–200 and E1A234–243 when transferred into LMP7+MECL-1-deficient mice. Taken together, our findings indicate that not absolute epitope quantities but early Ag-processing kinetics determine the ability of pathogen-derived peptides to elicit CD8 T cell responses, which is of importance for rational T cell vaccine design.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Most intracellular pathogen-derived proteins are degraded by host cell proteasomes, leading to the generation of antigenic peptides that associate with MHC class I molecules for presentation on the cell surface to surveilling CD8 T cells. It is well established that a variety of selective processes operating at the Ag processing and T cell levels limit the diversity of CD8 T cell responses mounted by the infected host (1, 2). For example, antigenic peptides must be liberated from their protein context, must be transported into the endoplasmic reticulum and must bind MHC class I molecules with high affinity. On the T cell site, precursors capable of recognizing the MHC-peptide combination must exist. Nevertheless, although multiple pathogen-derived peptides fulfill these criteria, only a few peptides are recognized during the acute phase of infection, suggesting that additional, thus far poorly understood, determinants influence the ability of single epitopes to dominate the CD8 T cell response.

Generation of most MHC class I-presented antigenic peptides requires proteasome activity. In infected tissues and professional APCs (pAPC),4 the constitutive catalytic proteasome subunits are replaced by three cytokine-inducible homologs (LMP2/induced (i)beta1, multicatalytic endopeptidase complex-like-1 (MECL-1)/ibeta2 and LMP7/ibeta5), resulting in the formation of so-called immunoproteasomes (3). Analyzing the products generated upon proteasome-mediated digestion of polypeptide substrates, we and others have found that the incorporation of the inducible subunits alters the cleavage preferences of proteasomes and accelerates the generation of a subset of CD8 T cell epitopes, which often dominate the pathogen-specific CD8 T cell response (4, 5). Nevertheless, immunosubunit incorporation mainly influences the frequency with which specific cleavage sites are used. Thus, immunoproteasomes generate specific epitopes rapidly and in high quantities; nevertheless, in many cases constitutive proteasomes excise the same epitopes when given enough time.

Because immunosubunit incorporation often does not lead to new peptide species, the reasons for the observed immunodominance of immunoproteasome-generated peptides are unclear. On the one hand, efficient immunoproteasome-mediated peptide liberation enhances the absolute densities of such peptides on priming pAPCs, possibly raising these over the threshold levels for CD8 T cell priming. In contrast, peptides that are efficiently processed will be the first to be presented on pAPCs with priming capacity. Such peptides may be more likely to be detected by Ag-specific naive T cells than peptides that are presented at a later time point, although by the same pAPC. In support of the last hypothesis, Willis et al. (6) recently showed that competition between CD8 T cells of different specificities for binding to the same dendritic cell (DC) is a very early event in T cell activation and restricts the number of T cells entering the response.

To determine whether the enhanced rate of processing and, consequently, a rapid presentation of immunoproteasome-dependent epitopes (4, 7) contributes to immunodominance, we have set up a well-controlled infection model in which previously defined immunoproteasome-dependent and independent epitopes are processed from the same Ag. This Ag is synthesized and secreted by the intracellular bacterium Listeria monocytogenes, excluding a role for defective ribosomal initiation products as a source of Ag in infected cells. Thus, in both infected cells and cross-presenting pAPCs, only mature, secreted bacterial Ags will enter the Ag-processing pathway. Processing of and CD8 T cell responses to different immunoproteasome-dependent and independent epitopes were analyzed in mice lacking the immunosubunits LMP7/ibeta5 and MECL-1/ibeta2 and compared with those in normal mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and infections

Recombinant L. monocytogenes (rLM)-OVA (8) and rLM-E1 were grown in brain-heart infusion medium (BD Biosciences), supplemented with 5 µg/ml erythromycin and 250 µg/ml spectinomycin, respectively, and harvested while in log phase. C57BL/6 (B6) mice were purchased from The Jackson Laboratory. LMP7+MECL-1 double-gene-deficient B6 mice (9) were maintained by in-house breeding under standard conditions. For primary infection, 6- to 12-wk-old female mice were inoculated i.v. into the tail vein with 0.1x LD50 rLM (5 x 103 rLM-E1 or 1 x 105 rLM-OVA) in 100 µl of PBS. Reinfection was performed 28–30 days later with 100-fold higher bacterial doses. All experiments involving animals were approved by the Institutional Committee on Animal Resources of the University of Rochester Medical Center (Rochester, NY).

Cell lines

EL4 and ANA-1 cells were cultured in RPMI (Invitrogen) and RMA-S cells in IMDM (Invitrogen Life Technologies), supplemented with 10% FCS (HyClone Laboratories), 2 mM L-glutamine, 30 µM 2-ME, and penicillin/streptomycin. E1A234–243-specific CD8 T cell line 5 (10), E1B192–200-specific CD8 T cell clone 100B6 (7), and an OVA257–264-specific CD8 T cell line (generated from the spleen of an rLM-OVA-infected B6 mouse) were cultured in IMDM containing 0.5 ng/ml human recombinant IL-2 (NCI Biological Resources Branch) and 50 µg/ml gentamicin and maintained by weekly restimulation with irradiated, peptide-pulsed LPS blasts. After five restimulations, 100% of OVA257–264-stimulated T cells expressed CD8 and were Ag specific.

Class I stability

RMA-S cells, lacking the TAP transporter, were seeded in the wells of 24-well plates at a concentration of 106 cells/ml (1 ml/well) in protein-free hybridoma medium (Invitrogen Life Technologies). Cells were incubated with 60 µM synthetic E1A234–243, E1B192–200 (both from Invitrogen Life Technologies) or without peptide at 37°C overnight and then harvested, washed three times with PBS, and chased at 37°C in the absence of peptide. Samples were taken after 0, 2, 4, and 6 h; washed with ice-cold PBS with 1% BSA and 0.02% NaN3; and stained for H2-Db or H2-Kb class I expression with biotin-conjugated mouse mAbs (28.14.8 and AF6–88.5 respectively) and PE-conjugated SA (eBioscience). Cells were analyzed on a FACSCalibur (BD Biosciences), using CellQuest software. Mean fluorescence levels detected on peptide-pulsed cells were corrected for the background levels, detected on cells that had been incubated minus peptide, and then used to determine MHC-peptide complex half-lives.

Intracellular cytokine staining

Approximately 10 x 106 erythrocyte-depleted splenocytes were incubated for 6 h with or without 500 nM synthetic peptide, in 1 ml of RPMI 1640 medium containing 50 µg/ml gentamicin and 9 µM monensin (eBioscience). Thereafter, cells were stained with an FITC-conjugated anti-mouse CD8 Ab (clone 53-6.7; eBioscience) in the presence of anti-CD16/32 (clone 2.4G2; eBioscience), fixed with 2% paraformaldehyde, and then stained with XMG1.2-PE (anti-IFN-{gamma}; eBioscience) in the presence of 0.5% saponin. Cells were analyzed on a FACSCalibur.

IFN-{gamma}-ELISPOT

Ninety-six-well MAIP ELISPOT plates (Millipore) were coated with 2 µg/ml AN18 (anti-mouse IFN-{gamma}) in 100 µl of PBS for at least 2 h at room temperature. Wells were then washed and blocked twice with RPMI 1640 medium. EL4 cells were pulsed with or without synthetic peptide (2 µM) for 2–3 h and then washed. Splenocyte dilutions, starting with 1 x 106 cells/well, were coincubated overnight with 3 x 104 unpulsed or peptide-pulsed EL4 cells in 100 µl of IMDM-medium with gentamicin. Thereafter, the plates were washed with PBS plus 0.01% Tween 20 (PBST), and IFN-{gamma} was detected by incubation with 2 µg/ml biotinylated XMG1.2, followed by 1 µg/ml alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch Laboratories), in PBST supplemented with 2% BSA. The assay was developed with the Vector AP substrate kit (Vector Laboratories). The plates were then washed, dried, and analyzed using the CTL ImmunoSpot scanner and software (Cellular Technology).

DC immunization

Bone marrow (BM)-derived DC of LMP7+MECL-1-deficient mice were expanded for 5 days with GM-CSF (20 ng/ml, derived from the culture supernatant of the cell line X63.Ag), activated overnight with 10 ng/ml LPS (Sigma-Aldrich) and then pulsed for 5 h with 10 µM synthetic E1A234–243, E1B192–200 or OVA257–264. Peptide-pulsed DCs were mixed and LMP7+MECL-1-deficient and B6 control mice were injected i.p. with 3 x 106 DCs in 100 µl of PBS. Seven days after immunization, spleens were harvested, and percentages of CD8 T cells reacting to the E1A234–243, E1B192–200, and OVA257–264 peptides (added at concentrations of 500 and 1 nM) were determined by intracellular IFN-{gamma} staining.

DC infection experiments

BM cells of LMP7+MECL-1-deficient and control B6 mice were cultured for 5 days with GM-CSF. CD11c+ cells (1 x 106) were plated in the wells of 24-well plates in RPMI 1640 medium without regular antibiotics but supplemented with 5 µg/ml erythromycin or 250 µg/ml spectinomycin and then infected for 1 h with rLM-OVA or rLM-E1, at multiplicities of infection of 1:250 and 1:100, respectively. After infection, the medium was replaced by RPMI 1640 medium without regular antibiotics but containing gentamicin. At different time intervals, intracellular bacterial growth and MHC class I cell surface trafficking were blocked by addition of tetracycline (20 µg/ml) and monensin (10 µM) to the culture medium. To detect processing and MHC class I presentation of bacterially secreted Ags, infected DCs and uninfected control DCs loaded with or without synthetic peptide were incubated overnight with 1 x 106 OVA257–264-, E1A234–243-, or E1B192–200-specific CD8 T cells. T cell activation was detected by intracellular IFN-{gamma} staining. This method has been shown before to reliably quantify peptide-MHC class I complexes presented on infected cells (11).

Splenocyte transfer

To deplete T cells, LMP7+MECL-1-deficient mice were injected i.p. with 200 µg of GK1.5 (anti-CD4) and 150 µg of 3.155 (anti-CD8) in 300 µl of PBS, on two consecutive days. At day 3, the mice were infected with 5 x 103 rLM-E1. After 36 h, spleens were harvested, pressed through a cell strainer, washed with PBS, filtered twice (40-µm pore size filter), and then injected i.v. into naive LMP7+MECL-1-deficient mice (1.5 donors to 1 recipient). Injected splenocytes contained <0.7% CD3+ cells. A second group of naive LMP7+MECL-1-deficient control mice received 5 x 103 rLM-E1 i.v. Both mouse groups were injected s.c. with 1 mg of ampicillin in 100 µl of PBS at the time point of rLM-E1 or splenocyte injection and received ampicillin through the drinking water (2 mg/ml), for 3 days. Seven days after infection, spleens were harvested and assayed for the presence of E1A- and E1B-specific CD8 T cells by IFN-{gamma} ELISPOT.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD8 T cell responses to the LM-secreted p60E1 Ag

To examine the mechanistic aspects underlying the preferential targeting of immunoproteasome-generated antigenic peptides, we modified L. monocytogenes to secrete a model Ag (p60E1) that is degraded by host cell proteasomes (12) and encompasses two well-studied model epitopes derived from the adenovirus type 5 early 1A and B regions (Fig. 1A) (7, 10). The two epitopes form stable complexes with the MHC class I H-2Db molecule (t1/2 H2-Db/E1Ap complex: 6.3 h; t1/2 H2-Db/E1Bp complex: 6.1 h; Table I) and can evoke vigorous CD8 T cell responses. Their immunodominance hierarchy alternates, depending on vector context (13, 14). One of the epitopes, E1B192–200, is processed rapidly and efficiently by immunoproteasomes, whereas constitutive proteasomes use the cleavage sites flanking this epitope with relatively low frequency (7, 10). The second epitope, E1A234–243, is processed efficiently in both the absence and presence of the immunoproteasome subunits (10). In p60E1, the two epitopes are surrounded by their natural flanking sequences (Fig. 1A) to conserve the natural epitope-surrounding proteasomal cleavage sites.


Figure 1
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  		FIGURE 1. Construction and characterization of rLM-E1. A, Sequences encoding the LM p60 promoter and p60 residues 1–360 were amplified and fused in frame to the E1 sequences E1A219–258 and E1B176–215 with the addition of a stop codon. Obtained p60-E1 construct was cloned into the pAT29 shuttle vector and introduced into L. monocytogenes (LM) strain 10403S. Approximately 75% of rLM-E1 retained pAT29p60E1 during 24 h of passage through B6 mice, indicating the stable expression of this plasmid. B, rLM-E1 secreted p60E1 into the medium, as detected by Western blot analysis on culture supernatant with an anti-p60 serum. C, rLM-E1 infection induced LLO296–304-, E1A234–243-, and E1B192–200-specific T cell responses in B6 mice, as measured by IFN-{gamma} ELISPOT. Bars, means (n = 5) ± SEM.

 

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  		Table I. Stability of MHC class I-rLM peptide complexes

 
The constructed Listeria strain rLM-E1 (Fig. 1A) secreted the heterologous p60E1 protein (Fig. 1B). To determine whether rLM-E1 elicited E1-specific CD8 T cell responses, B6 mice were inoculated i.v. with a sublethal bacterial dose and responses in the spleen were quantified 7 days later (Fig. 1C). rLM-E1-infected mice responded most vigorously to E1B192–200, whereas E1A234–243 and a L. monocytogenes-derived epitope (LLO296–304) elicited smaller CD8 T cell responses. Uninfected mice failed to react to any of these epitopes (not shown). Thus, E1B192–200 dominates over E1A234–243 when processed from p60E1.

rLM-E1-infected immunosubunit-deficient mice fail to respond to E1B192–200

To examine whether immunoproteasomes played a role in the priming of E1B-specific CD8 T cells, we infected LMP7+MECL-1 double-gene-deficient B6 mice (9) and control mice with sublethal doses (0.1 x LD50) of rLM-E1 or rLM-OVA (8), which secretes truncated OVA encompassing the immunoproteasome-independent (15) OVA257–264 epitope. Quantitation of CD8 T cell responses to the different model epitopes at day 7 after infection (Fig. 2A) showed that rLM-E1-infected LMP7+MECL-1-deficient mice failed to respond to the immunoproteasome-dependent E1B epitope, upon both primary and recall infection, whereas E1B192–200 was the predominant target of the E1-specific CD8 T cell response in rLM-E1-infected control B6 mice. Frequencies of CD8 T cells responding to the p60E1-derived E1A control epitope were similar between the two mouse groups (Fig. 2A). Moreover, both B6 control and gene-deficient mice responded vigorously to rLM-OVA-derived OVA257–264 (Fig. 2A). Thus, mice lacking LMP7+MECL-1 were unable to respond to p60E1-derived E1B192–200 but mounted normal responses to the p60E1-derived E1A234–243 and OVA-derived OVA257–264 control epitopes.


Figure 2
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  		FIGURE 2. Lack of E1B192–200 responses in LMP7+MECL-1-deficient mice. LMP7+MECL-1 gene-deficient and control B6 mice were infected i.v. with primary doses of 5 x 103 rLM-E1 or 1 x 105 rLM-OVA, or secondary doses of 5 x 105 and 1 x 107 bacteria, respectively (A), or injected i.p. with E1B192–200-pulsed or unpulsed DC (B). Primary responses to the indicated peptides were measured on day 7 (A and B), recall responses (A) on day 5 after rLM or DC injection by intracellular IFN-{gamma} staining. Values are mean results for individual mice (A and B) ± SEM (B). Similar results were obtained in five independent experiments. LM, L. monocytogenes; wt, wild type; w/o pep., without peptide.

 
To test whether the absence of E1B192–200-specific responses in rLM-E1-infected LMP7+MECL-1-deficient mice could be explained by defects in the TCR repertoire, mice were immunized with GM-CSF-expanded BM-derived DCs pulsed with synthetic E1B192–200. Both LMP7+MECL-1-deficient and control mice mounted E1B192–200-specific responses (Fig. 2B). Most of the responding CD8 T cells reacted to both high (500 nM) and low (1 nM) concentrations of synthetic E1B192–200 (Fig. 2B), indicting that both gene-deficient and control mice possess naive T cells with high affinity for the Db/E1B192–200 complex. Thus, although we cannot exclude that the E1B-specific T cell repertoire may differ between control and gene-deficient mice, our data show that the defective priming of E1B-specific responses in rLM-E1-infected LMP7+MECL-1-deficient mice cannot be explained by a lack of T cell precursors.

Delayed presentation of E1B192–200 by rLM-p60E1-infected, immunosubunit-deficient DCs

To further explore the relationship between Ag-processing efficiency and CD8 T cell priming, we determined whether the absence of LMP7+MECL-1 altered the presentation of the different model epitopes by GM-CSF-expanded BM DC infected with rLM-E1 or rLM-OVA (Fig. 3B). Bacterial protein synthesis and MHC class I cell surface transport were stopped at different time points after infection by the addition of tetracycline and monensin, respectively, and epitope presentation was detected using established CD8 T cell lines that displayed similar sensitivities for their respective peptide ligands (Fig. 3A). These analyses of epitope presentation (Fig. 3B) revealed that rLM-E1-infected DC of LMP7+MECL-1-deficient and control B6 mice presented the E1A epitope with similar kinetics; i.e., E1A234–243 slowly accumulated on the surface of both types of DCs. In contrast, the amounts of E1B peptide increased rapidly on B6 DCs (first detectable after 3 h of infection with maximal T cell activation reached after 5 h (Fig. 3B) but not on LMP7+MECL-1-deficient DC, which needed a longer period of time to accumulate detectable amounts. The control rLM-OVA-derived OVA257–264 epitope was rapidly presented by both B6 and gene-deficient DC (Fig. 3B). Comparing the results of the kinetic analyses of epitope presentation with the sizes of epitope-specific CD8 T cell responses in Fig. 2, we find a remarkable correlation between rates of epitope presentation and immunogenicity of the respective model epitopes in B6 (E1B > E1A) and LMP7+MECL-1-deficient mice (E1A > E1B).


Figure 3
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  		FIGURE 3. Delayed E1B192–200 presentation on infected LMP7+MECL-1-deficient BM-DC. A, OVA257–264-, E1A234–243-, and E1B192–200-specific CD8 T cells incubated with dilutions of synthetic peptides, loaded on ANA-1 cells, recognize their respective ligands with comparable sensitivities. T cell activation was measured by intracellular IFN-{gamma} staining. B, Presentation kinetics of p60E1-derived E1B192–200 and E1A234–243 and of OVA-derived OVA257–264 on infected BM-DC. At different time points after infection with rLM-E1 or rLM-OVA, intracellular bacterial growth and MHC class I cell surface trafficking were blocked with tetracycline and monensin. Presentation of the different antigenic peptides was detected with the CD8 T cells shown in A. Results are representative of three experiments. wt, Wild type.

 
Kinetics of presentation determine the immunogenicity of E1B192–200

Based on our data thus far, the inability of infected LMP7+MECL-1-deficient mice to respond to p60E1-derived E1B192–200 may be explained by the delayed processing and presentation of this epitope in the absence of immunoproteasomes. Alternatively, it is possible that the epitope quantities presented on DC of these mice are insufficient to activate naive E1B192–200-specific CD8 T cells. The initiation of both primary and recall CD8 T cell responses requires Ag presentation by DC (16, 17). To examine first whether DCs of infected mice present E1B in vivo, LMP7+MECL-1-deficient and control B6 mice were immunized with E1B192–200-pulsed cells. After 28 days, the immune mice were challenged with rLM-E1 and E1B-specific CD8 T cell responses were quantified in the spleens at day 5 after infection. rLM-E1 induced vigorous E1B-specific CD8 T cell responses in both control and LMP7+MECL-1-deficient DC+E1B192–200 immunized mice (Fig. 4A) whereas, at this time point, no or small E1B-specific CD8 T cell responses were detectable in the spleens of LMP7+MECL-1-deficient and control B6 mice that had been immunized with non-peptide-pulsed DCs. These findings indicate that E1B192–200 is processed from p60E1 and presented on DCs of rLM-E1- infected mice.


Figure 4
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  		FIGURE 4. In vivo presentation of E1B192–200 in LMP7+MECL-1-deficient mice. A, E1B192–200-specific CD8 T cells, primed by immunization with E1B192–200-pulsed DC, expanded upon reinfection with rLM-E1. B, Splenocytes of T cell-depleted LMP7+MECL-1 gene-deficient mice, infected for 36 h with rLM-E1-, primed E1B192–200-, and E1A234–243-specific CD8 T cells in naive LMP7+MECL-1–/– mice, as measured by IFN-{gamma} ELISPOT. Similar results were obtained in three independent experiments. wt, Wild type; prim., primary; pept., peptide; sec., secondary; bars, SEM.

 
Previous studies have shown that the first 24 h of infection are crucial for the priming of L. monocytogenes-specific CD8 T cells (18). To discern whether the quantities or kinetics of E1B192–200 presentation explain the inability of rLM-E1-infected LMP7+MECL-1-deficient mice to mount E1B192–200-specific responses, we determined whether pAPC of infected LMP7+MECL-1- deficient mice that present the rLM-E1-derived epitopes at steady state levels are able to prime E1B-specific CD8 T cell responses in naive gene-deficient mice. LMP7+MECL-1-deficient mice were depleted of CD4 and CD8 T cells to prevent the formation of clusters between Ag-presenting pAPC and T cells and were then infected with rLM-E1. After 36 h, splenocytes were harvested and transferred into naive LMP7+MECL-1-deficient mice. The recipient mice were simultaneously treated with ampicillin, to avoid infection with donor-derived bacteria. Quantitation of CD8 T cell responses seven days later by IFN-{gamma} ELISPOT (Fig. 4B) revealed significant numbers of E1B-specific CD8 T cells in the spleens of the recipient mice, which equaled the numbers of E1A-specific CD8 T cells. No E1-specific responses were detected in control ampicillin-treated LMP7+MECL-1-deficient mice that, instead of receiving splenocytes, had been infected with rLM-E1, implying that the E1-specific responses in splenocyte recipient mice were unlikely to be evoked by surviving, donor-derived rLM-E1. From these data, we infer that the quantities of E1B192–200 processed and presented by pAPC of infected LMP7+MECL-1 deficient mice are sufficient to prime naive CD8 T cells and, furthermore, that the two p60E1-derived epitopes are codominant when offered in a preprocessed form. Our results therefore strongly suggest that the kinetics of epitope appearance on single pAPC rather than solely epitope quantity may explain for the immunogenicity or lack of certain antigenic peptides.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Taken together, our data demonstrate a new mechanism that controls CD8 T cell priming on the level of Ag processing. Incorporation of the IFN-{gamma}-inducible subunits into proteasomes leads to a more frequent cleavage of specific peptide bonds and thereby 1) to a faster appearance of certain peptides on the pAPC surface and 2) to enhanced peptide quantities. Different from the general assumption (19), we here show that the effects of immunoproteasomes on epitope presentation kinetics are more important than their effects on overall epitope quantity.

How can one explain our finding that rates of processing influence the immunogenicity of CD8 T cell epitopes? Multiple studies have indicated that Ag presentation by DCs is highly regulated. Thus, their capacity to capture, process, and present Ags, including those presented by MHC class I molecules, depends on the maturation status of the DC. Hence, it is possible that DCs have a limited time window only to process and present Ag and to engage in stable, long-lasting and probably monogamous interactions (20) with naive CD8 T cells that scan the draining lymph nodes for pAPCs presenting the appropriate peptide. In addition, previous studies have shown that the number of pAPCs presenting pathogen-derived peptides in the lymph node is only small and that these pAPCs are short-lived (21). Therefore, it is tempting to speculate that the availability of pAPC presenting pathogen-derived Ags represents the bottle neck by which the immunodominance hierarchy of the elicited CD8 T cell response is determined. An epitope that is presented earlier on pAPCs has a greater chance of binding naive CD8 T cells than an epitope that is presented later. Once a stable interaction is formed, it probably is less likely for the pAPC to form additional stable interactions with other naive T cells. Therefore, an epitope that is presented early on the pAPC would in this way prevent naive CD8 T cells specific for epitopes presented at later time points from being primed, leading to the previously (2) postulated immunodomination of in this case early presented epitopes. Thus, the MHC-encoded immunoproteasome subunits could use such a mechanism to convert cryptic epitopes into dominant epitopes by accelerating the generation and, thereby, the presentation of specific peptides on pAPCs. In good agreement with this model, Willis et al. (6) recently demonstrated that competition between T cells of different specificities for interaction with the same Ag-presenting DC is a very early event in T cell activation, which occurs within the first 5 h of immunization and affects the number of T cells entering the response.

Our observation that the time point of epitope appearance on pAPCs plays an important role in immunogenicity is in line with findings by others that emphasize that the events occurring very early after infection are of crucial importance for the priming of T cell responses (6, 18, 22). For example, Pamer and coworkers (18) found that the first 24 h of infection with L. monocytogenes are decisive for the initiation of Ag-specific CD8 T cell responses. Furthermore, immunologists studying the fine specificity of CD8 T cell responses to several murine and human viral pathogens have shown that CD8 T cells frequently respond to peptides that derive from early expressed proteins (23, 24). Nevertheless, the mechanisms underlying the preferential targeting of early expressed vs rapidly processed epitopes may differ. In case of viral infections, the individual epitopes localize on different viral Ags that are expressed at different time points after infection and probably presented by different pAPCs. In contrast, the two E1 epitopes derive from the same Ag and thus are processed by the same pAPC but with differing efficiencies and therefore presented at differing time points after infection.

Many studies aimed to design vaccines that induce protective CD8 T cell-mediated immunity use vector organisms to introduce the relevant Ags. Our finding that rates of epitope generation from vector-encoded Ags influence the immunogenicity of CD8 T cell epitopes implies that such vaccines may be more effective when rapid processing of an included epitope is guaranteed. Thus, future studies identifying sequences that allow rapid proteasome-mediated epitope liberation may support the development of more efficient T cell-based vaccines.


   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 the Howard Hughes Biomedical Research Support Program for Medical Schools and National Institutes of Health Grant AI064576 (to A.S.) and by the German Research Council (DFG Za 280 to D.Z.). Back

2 P.D. and D.M.W.Z. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. A. Sijts, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, The Netherlands. E-mail address: E.J.A.M.Sijts{at}vet.uu.nl Back

4 Abbreviations used in this paper: BM, bone marrow; DC, dendritic cell; LMP, low-molecular-mass polypeptide; MECL-1, multicatalytic endopeptidase complex-like-1; pAPC, professional APC; rLM, recombinant Listeria monocytogenes; i, induced. Back

Received for publication January 10, 2007. Accepted for publication January 10, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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