Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Immunol. Author manuscript; available in PMC 2010 April 15.
Published in final edited form as:
PMCID: PMC2756161

The amino acids sequences flanking an antigenic determinant can strongly affect MHC class I cross-presentation without altering direct-presentation1


Direct presentation (DP) and cross presentation (CP) on MHC I by professional antigen presenting cells are defined by the internal or external source of the Ag, respectively. While some Ags are substrates for both DP and CP, others are only substrates for DP. The reasons for this difference remain largely unknown. Here we studied in tissue culture and also in vivo, the effects of altering the length and sequence of the amino acid chains flanking an MHC class I restricted determinant (the chicken ovalbumin OVA 258–265, SIINFEKL) that is normally a good substrate for both DP and CP. We demonstrate that CP but not DP strictly requires flanking N and C terminal extensions of minimal length. Furthermore, we show that removal but not replacement of just one amino acid 22 residues downstream from the determinant is sufficient to strongly affect CP without affecting either protein stability or DP. Thus, our work shows that the flanking residues of an antigenic determinant can differentially affect CP and DP, and that features of the Ag other than half-life can have a major impact in CP. Our studies may have implications for understanding CP in viral infections and possibly for the design of new vaccines.

Keywords: Animals-Rodent, Infections-Viral, Molecules-Antigens/Peptides/Epitopes, Molecules-MHC, Processes-Antigen Processing/Presentation


CD8+ T lymphocytes (TCD8+) have an essential role in the control of many viral infections, intracellular bacteria and cancer. In the case of viruses, TCD8+ recognize viral peptides, 8–10 amino acids (AA) in length, bound to MHC I molecules (14). For most cells, the viral peptides bound to MHC I are derived from the proteasomal degradation of viral proteins synthesized by the infected cell in a process known as direct presentation (DP). However, bone marrow-derived professional antigen presenting cells (pAPC), which are essential to initiate TCD8+ responses from naïve precursors (5, 6), can also present antigens acquired from other cells. This process, widely known as cross presentation (CP) (79), is thought to be important to initiate TCD8+ responses to pathogens that do not infect pAPC or to those that infect pAPC but cripple their antigen presenting functions (5, 911). CP is also thought to be mainly responsible for TCD8+ responses to tumors (12). Therefore, understanding the rules of CP might also be important for the development of novel vaccines. For example, there is evidence that cross presentation is required for the generation of TCD8+ responses to DNA vaccines (1315).

While it is clear that determinants derived from well-defined Ags such as purified proteins bound to beads, inactivated viruses, and virus-like-particles can be presented by pAPC in vitro and in vivo (11), we still know very little about the structural characteristics required for efficient physiological CP when the Ag is expressed by Ag donor cells. Previously, we showed that the Kb-restricted determinant SIINFEKL (AA single letter code) from chicken ovalbumin (OVA, 386 AA) was cross-presented better when the precursor was synthesized in vaccinia virus (VACV)-infected donor cells as a cytosolic fragment than as the full-length OVA (16). Importantly, we also showed that OVA 258–265 synthesized as the minimal determinant, i.e., without N or C-terminal extensions, is not a substrate for CP. Conversely, expression of the minimal determinant alone results in stronger DP than with any other precursor. This important observation, later confirmed by others (17), suggested that CP does not result from the transfer of the minimal determinant either free or associated with chaperones although an exception was recently found (18). In this report, we further use OVA as a model to explore the structural requirements for efficient CP of Ags donated by infected cells and compare them with the requirements for DP as a way of testing the hypothesis that the rules that govern CP and DP are different. Our results demonstrate that CP but not DP requires flanking AA sequences of minimal length at both ends of the determinant. Of interest, the negative effect of shortened flanking chains in CP did not necessarily reflect decreased protein stability. Together, our findings provide novel insights towards understanding the mechanisms whereby TCD8+ responses are induced. Furthermore, our work may have important implications for the rational design of new vaccines.

Materials and Methods

All experiments were repeated a minimum of three times with similar results. Each figure depicts a representative experiment.

Cell lines

All cells were maintained in RPMI 1640 medium supplemented with 10% FCS (Atlanta Biologicals, Atlanta, GA), 2mM L-glutamine, penicillin-streptomycin, 0.01M HEPES buffer, and nonessential amino acids (all from Invitrogen, San Diego, CA) and 5 ×10−5 M 2-ME (Sigma-Aldrich, St. Louis, MO) (RPMI-10). A9 cells (ATCC no. CCL-1.4; American Type Culture Collection, Manassas, VA) are a derivative of the strain L (H-2K). A9-T7 cells (19, 20) are A9 cells stably transfected with T7 polymerase (a gift from B. Moss, National Institute of Allergy and Infectious Diseases, Bethesda, MD). MC57G cells (ATCC no. CRL-2295) are a C57BL/6-derived fibrosarcoma (H-2b). B3Z (a gift from N. Shastri, University of California, Berkeley, CA) is a TCD8+ hybridoma that produces β-galactosidase (β-gal) upon recognition of the OVA 258–265 in the context of the H-2Kb molecule (21) without the need of costimulation. All cells were grown at 37°C in an atmosphere of 5% CO2.


All constructs were generated by PCR and recombinant PCR using Pfu polymerase (Clontech) and appropriate primers. All 5’ primers contained a restriction endonuclease cloning site (in most cases BamH I), a Kozak sequence (CACC) and an artificial translation-initiation ATG (when not naturally present) coding for an initial Met that is presumably cotranslationally removed. This was followed by sufficient nucleotides of the sequence of interest to provide for a predicted Tm of 60°C when annealed with the template. The 3’ primer also contained sufficient sequence for a predicted Tm of 60°C, as well as an artificial TAA termination codon (when not naturally present) and a restriction endonuclease cloning site (in most cases Not I). All constructs were cloned into plasmid Bluescript II SK+ using BamHI and Not I restriction enzymes in most cases. Sequences were confirmed by bi-directional sequencing using T7 and T3 primers and additional internal primers when necessary. Throughout the paper we refer to the constructs as X-SIINFEKL-X where X represents the number of AA that precede or follow SIINFEKL. Unless indicated, these are the natural OVA AAs.


C57BL/6 mice were bred at Fox Chase Cancer Center’s Laboratory Animal Facility or purchased from NCI-Frederick. All experiments were performed with males 6–8 weeks of age. All experiments were performed under protocols approved by the Institutional Animal Use and Care Committee.


Wild type WR and T7 polymerase recombinant vaccinia virus (VACV, a gift from B. Moss) were produced and stored as before (22). The VACV recombinants 61-SIINFEKL-121, 61-SIINFEKL-0 and 46-SIINFEKL-16 were produced and selected based on plaque size exactly as described by Blasco and Moss (23)

Direct presentation assays

For DP by MC57G cells 2 × 105 cells were plated in 24-well plates, incubated overnight, infected for 1 h with recombinant VACV-T7 (10 PFU/cell), and transfected with the indicated constructs using lipofectamine 2000 (Invitrogen) as recommended by the manufacturer. Four hours later, the cells were harvested, fixed with 0.5% paraformaldehyde in PBS, washed, and varying numbers added in duplicate to 96-well plates. B3Z responder cells (105) were added to each well. Plates were incubated for additional 18–24 h to allow for the activation of the hybridoma. β-gal activity was determined in lysates of the cells using the Galactostar chemiluminescent kit (Tropix; Applied Biosystems, Foster City, CA) as described (24). For DP by Mϕ, bone marrow Mϕ were generated as previously described (24) and 106 were infected in tubes with 10 PFU of the indicated recombinant VACV. After 2 h, the cells were washed, placed in 96-well plates in different concentration and incubated with 105 B3Z cells overnight. β-gal activity was determined as for MC57G cells.

Cross-presentation assays

Ag donor cells were A9-T7 which are a C3H (H-2k) mouse fibroblast cell line (A9 cells; ATCC no. CCL-1.4) that stably expresses the phage T7 polymerase. These cells cannot present SIINFEKL on Kb because they do not express this H-2 molecule. Also, when uninfected A9-T7 cells are transfected with plasmids encoding polypeptides controlled by the T7 promoter, they do not express them because the uncapped RNA is very unstable. However, when the transfected A9-T7 cells are also infected with VACV, the RNA is stabilized resulting in the production of large amounts of protein. In our assays, A9-T7 were seeded in six-well plates (6×105 cells/well) or 24-well plates (2×105 cells/well) incubated overnight at 37°C, and transfected with the indicated plasmids using lipofectamine 2000 as per the manufacturers instructions. Four hours later, the cell monolayer was washed twice with PBS and the cells were infected with 10 PFU/cell of wild-type VACV in medium containing 2.5% FCS. Following 1-h incubation, the cell monolayer was washed twice with complete medium and incubated in complete RPMI 1640 for 16 hours. When required, the donor cells were incubated with 2 µM lactacystin as previously described (16) for 2 h.

Donor cells were rinsed and 4 ×105 Mϕ as pAPC were added to each well and incubated for 4-h. In our assay, B6 macrophages (Mϕ) can present SIINFEKL on Kb because they express this molecule. However, they cannot express the Ag even if they become infected, because they are not transfected nor do they express the T7 polymerase. The Mϕ/donor cell mixtures were harvested with a rubber policeman, washed, and varying numbers were added to duplicate wells of 96-well plates. B3Z cells (105) were added to each well and their activation was determined as for DP. No cross-presentation was observed when we used parent A9 cells as mock donor cells subjected to identical treatment as donor A9-T7 (not shown).

Pulse-chase experiments

A9-T7 cells were transfected and infected in 6 well plates as for the cross-presentation assay. 16–20 h post infection, the cells were washed twice with PBS and 1ml labeling media (Methionine-deficient RPMI 1640 (Sigma), 2% dialyzed FCS, 50 µCi/ml 35S M/C (Tran35S-label, MP Biomedicals) was added. After incubating for 10 min at 37 °C, the labeling media was aspirated and the monolayer was washed twice by adding 2 ml ice cold RPMI-10 followed by aspiration. After aspirating the last wash media, 1 ml ice cold RPMI-10 was added to each well, the cells were harvested using a rubber policeman and aliquoted into microcentifuge tubes. One aliquot (time 0) was immediately centrifuged at 4°C, the supernatant aspirated, and the pellet frozen at −80 °C. The other aliquots were placed in a water bath at 37 °C, incubated for the indicated times and then treated as above. After all samples were collected and frozen, the pellets were thawed on ice, vortexed, and 40 µl of SDS-PAGE loading buffer (BioRad) containing 2-ME were added. The cells in loading buffer were vortexed, incubated on ice for 15 min, heated for 5 min at 98 °C, centrifuged at high speed, and 20 µl were loaded into pre-cast gels (BioRad). Proteins were electrophoresed (100 V, 4 °C) and transferred to a nitrocellulose membrane (BioRad) (100 V, 60 min) using a Mini-protean II apparatus. Radioactivity was visualized by directly exposing BioMax film (Kodak) to the membrane for 12–72 h.

In vivo cross-priming

Cells from the mesenteric, axilar, inguinal and popliteal lymph nodes of Rag-1 deficient, Thy1.1 congenic, OT-1 transgenic mice were pooled, made into single cell suspensions, stained with CFSE essentially as described (25, 26) and 106 were injected intravenously into C57BL/6 recipients. One day later, donor cells prepared exactly as for the in vitro assay were inoculated subcutaneously in the left thigh of the OT-1-Thy1.1 recipients. Five days later, mice were euthanized, the popliteal lymph nodes collected, made into single cell suspensions, and stained with anti CD8-PE-Cy7 and anti-Thy1.1-PE antibodies (both from Pharmingen, Becton Dickinson). Data collection was gated on TCD8+ and 200,000 events were acquired. The data was analyzed with FlowJo software.


CP but not DP of SIINFEKL requires flanking sequences at both ends of the determinant

We have recently shown that during VACV infection, expression of the minimal determinant SIINFEKL (see materials and methods for construct nomenclature) results in stronger DP than expression of an N-terminally truncated form of OVA that remains in the cytosol. In this construct SIINFEKL is preceded by 61 and followed by 121 AA (61-SIINFEKL-121). However, SIINFEKL was not a substrate for CP while 61-SIINFEKL-121 was cross-presented with high efficiency (16), indicating that CP does not involve the transfer of the fully processed Ag from donor cells to pAPC. From basic and applied standpoints, it is of interest to discover the rules that determine whether Ags are substrates for CP and how this may differ for DP. Since 61-SIINFEKL-121 was cross-presented with high efficiency while SIINFEKL was not, it was possible that CP required the determinant to be extended at the C and/or N terminus. To test this, we made constructs 61-SIINFEKL-0 and 0-SIINFEKL-121 lacking C- or N terminal extensions. Unlike control 61-SIINFEKL-121 which was cross-presented with high efficiency, neither 0-SIINFEKL-121 nor 61-SIINFEKL-0 were substrates for CP (Figure 1A). On the other hand, both 61-SIINFEKL-0 0-SIINFEKL-121 were good substrates for DP (Figure 1B) albeit 0-SIINFEKL-121 was presented with somewhat decreased efficiency as compared to control 61-SIINFEKL-121 most likely due to the easy accessibility of the determinant to the destructive action of cytosolic and ER amino peptidases (27, 28). In additional experiments, we found that other constructs lacking C-terminal extensions such as 13-SIINFEKL-0 and 3-SIINFEKL-0 were substrates for DP but not for CP (not shown). Therefore, distinct from DP, SIINFEKL must be flanked by extensions at both the N and C terminal ends for efficient CP.

Figure 1
CP but not DP of SIINFEKL requires flanking sequences at both ends of the determinant

Optimal CP of SIINFEKL requires an N-terminal extension longer than 35 AA

We next determined the minimal length of the N-terminal extension. Because 61-SIINFEKL-121 was a substrate of CP while 0-SIINFEKL-121 was not, we made constructs with N-terminal extensions of various lengths and the C-terminal extension fixed at 121 AA long. We found that constructs with relatively long N-terminal extensions such as 61-SIINFEKL-121, 58-SIINFEKL-121 and 46-SIINFEKL- 121 were excellent CP substrates, while 18-SIINFEKL-121 and 13-SIINFEKL-386 were cross-presented poorly (Figure 2A). 35-SIINFEKL-121 (not shown) fluctuated between experiments within the values of maximal and low CP while 1-SIINFEKL-121 and 2-SIINFEKL-121 were not cross-presented at all (not shown). All these constructs, however, were excellent substrates for DP (Figure 2B and data not shown) indicating that they were expressed at similar levels and are good substrates for the processing machinery of infected cells. Thus, changes in the length of the N-terminal extension do not have major effects on DP but can profoundly affect the CP of SIINFEKL. Although the exact length requirement for the N-terminal extension was not determined, that CP of 35-SIINFEKL-121 was cross-presented with variable efficiency while 46-SIINFEKL-121 was presented with high efficiency indicates that for the natural OVA sequences optimal CP requires constructs with N-terminal extensions longer than 35 AA.

Figure 2
Optimal CP of SIINFEKL requires substrates with N-terminal extensions longer than 35 AA

Optimal CP of SIINFEKL requires C-terminal extensions longer than 23 AA

Next, we examined the length and sequence requirements for the C-terminal extension. Because 46-SIINFEKL-121 was cross-presented efficiently (Fig. 2A), we made new constructs with the N-terminal extension fixed at 46 AA long and progressive deletions of the C-terminal extension. Although some non-reproducible inter-experimental variations were observed, we found that 46-SIINFEKL-121, -85, -60, -55, -50, -45, -40, -35 -30 (not shown) and -25, -24, -23 and -22 were cross-presented with relatively similar efficiency as control 61-SIINFEKL-121 but CP of 46-SIINFEKL-21 was dramatically and reproducibly reduced ~60–80% (Figure 3A). Constructs with even shorter C-extensions such as 46-SIINFEKL-16, -11, -6 and -0 were not substrates for CP (Figure 3A and data not shown). On the other hand, all these constructs were excellent substrates for DP (Figure 3B and data not shown). Thus, CP but not DP of SIINFEKL requires a minimal C-terminal extension of a least 22 AA. The importance of the C-terminal extension in CP is dramatically exemplified by the difference in CP between 46-SIINFEKL-21 and 46-SIINFEKL-22 which differ in just 1 AA. Interestingly, similar to our previous report for 61-SIINFEKL-121, the CP of 46-SIINFEKL-21 was completely abolished when the donor cells were exposed to the proteasome inhibitor lactacystin (Figure 4). Thus, the proteasome of donor cells plays a major role in CP even for a construct of minimal size.

Figure 3
Optimal CP of SIINFEKL requires C-terminal extensions longer than 23 AA
Figure 4
Inhibition of the donor cell proteasome blocks CP of the smallest cross-presented construct

Changes in the C-terminal extension do not affect DP presentation by Mϕ

In the experiments above the Ag was synthesized by fibroblast cell lines (i.e. A9-T7 for CP and MC57G for DP). Yet, only for CP the APCs were Mϕ. Thus, we compared DP by Mϕ of 61-SIINFEKL-121 (which is cross-presented), and 61-SIINFEKL-0 and 46-SIINFEKL-16 (which are not cross-presented). Because primary Mϕ are difficult to transfect in this case we used recombinant VACV encoding the constructs themselves. As shown in Figure 5, Mϕ were highly efficient for the DP of the three constructs. This result rules out the possibility that Mϕ selectively fail to process and present some constructs.

Figure 5
Changes in the C-terminal extension do not affect DP presentation by Mϕ

Variable effects on CP of SIINFEKL as a result of changes in the sequence of the N- and/or C- terminal extensions

The data thus far shows that the length of the N- and/or C-terminal extension profoundly affects CP but not DP of SIINFEKL. We next tested whether the natural OVA sequences were required. We replaced the 46 N-terminal AA in 46-SIINFEKL-121 with identical length sequences from the N terminal end of green fluorescent protein (GFP) to generate 46(N-GFP)-SIINFEKL-121 or from the C terminal end of influenza virus nucleoprotein (NP) to generate 46(C-NP)-SIINFEKL-121. Results showed that 46(N-GFP)-SIINFEKL-121 was as good substrate for CP as 61-SIINFEKL-121 while 46(C-NP)-SIINFEKL-121 was a very poor CP substrate (Figure 6A). On the other hand, the three constructs were equally suitable substrates in DP assays (Figure 6B). We also replaced the C-terminal 121 AA in 46-SIINFEKL-121 and 46-SIINFEKL-21 with identical length sequences from the N-terminus of GFP (46-SIINFEKL-121(N-GFP), which was an excellent substrate for CP and DP, and 46-SIINFEKL-21(N-GFP) which was a substrate for DP but not for CP (Figure 6 C and D). We also found that 46-SIINFEKL-0 fused to full length GFP (46-SIINFEKL-GFP) was an excellent substrate for both presentation pathways. In additional experiments we found that changing the original K at the last position in OVA 46-SIINFEKL-22 (46-SIINFEKL-21K) for a conservative (46-SIINFEKL-21R), a neutral (46-SIINFEKL-21A) or a non-conservative (46-SIINFEKL-21D) residue did not affect either CP or DP (not shown). Altogether, these results confirm the length requirement at the C-terminus of SIINFEKL and demonstrate that CP of SIINFEKL does not have strict requirements for specific sequences at either the N- or C-terminal extension (because at both ends the native OVA sequences could be replaced for those of GFP), but that particular sequences (such as those of NP at the N-terminus) can have very deleterious effects.

Figure 6
Variable effects on CP of SIINFEKL as a result of changes in the sequence of the N- and/or C- terminal extensions

Low CP of 46-SIINFEKL-21 is not due to a short half-life

Work by Norbury et al. using a construct consisting of NP-SIINFEKL-GFP concluded that protein half-life is a major determining factor of efficient CP (17). In this model, proteins with short half-life are presented inefficiently because the amount of protein available for transfer between donor cell and pAPC is limiting. We therefore tested whether the difference in CP between some of our constructs could also be explained by different half-life. However, we did not detect a decrease in the stability of 46-SIINFEKL-21 as compared to 46-SIINFEKL-22 or 246-SIINFEKL-25 (Figure 7) or 61-SIINFEKL-121 (not shown) as determined by changes of the intensity of the corresponding bands in a 2-h pulse-chase experiment with 35S M/C followed by SDS-PAGE electrophoresis, transfer to a membrane and non-saturating film exposure. Indeed, all these proteins appeared to be very stable because their signals did not decrease substantially during the 2-h chase. From this experiment we conclude that the profound decrease in CP that we observed in 46-SIINFEKL-21 as compared to 46-SIINFEKL-22 and 46-SIINFEKL-25 and 61-SIINFEKL-121 is not due to a decrease in protein stability.

Figure 7
The reduced CP of 46-SIINFEKL-21 is not due to a shorter half-life

The rules of in vitro CP also apply to in vivo cross-priming

Because it is possible that the rules of CP in vitro may not be applicable in vivo, we extended our experiments to an in vivo setting. Ag donor cells were prepared exactly as for the in vitro assay and 106 were injected subcutaneously into the flanks of C57BL/6 mice that had been adoptively transferred with CFSE labeled, Kb-SIINFEKL-specific, transgenic OT1-Thy 1.1 T cells (29). Using this approach, we compared the efficiency of CP for various constructs by analyzing the expansion and proliferation of OT1 cells in the popliteal lymph node draining the site of immunization four days after immunization. We found that 46-SIINFEKL-16 did not induce proliferation above background levels and, although somewhat more efficient than in the in vitro assay, the CP of 46-SIINFEKL-21 was clearly lower than that of longer constructs such as 46-SIINFEKL-22, and 46-SIINFEKL-25 (Figure 6) or 46-SIINFEK-55 and 61-SIINFEKL-121 (not shown). It should also be noted that control mice that received A9 cells (that do not have the T7 polymerase and, thus, do not express Ag) infected with VACV and transfected with OVA 197–386 did not proliferate over background (not shown) confirming that our assay measures CP and not Ag expression by the pAPCs that may have picked-up the plasmid. Therefore, the rules of in vitro and in vivo CP of SIINFEKL are comparable.


CP of Ags on MHC I molecules is a well-established phenomenon that has been demonstrated both in vitro and in vivo (11). However, to what extent CP contributes to overall TCD8+ responses is hotly debated (9, 30, 31), and this is partly due to the fact that CP could not be demonstrated for some antigens that, nonetheless, induce strong TCD8+ responses (32, 33). Work by Norbury et al. has shown that the cross-presentation of a model Ag was lost if rapidly degraded within the donor cell (17). In addition, Wolkers et al. showed that two different determinants were cross-presented when they localized to the mature domain of recombinant protein but not to the signal sequence, which is normally rapidly degraded. On the other hand, DP was not affected by the localization of either determinants to one or the other region of the protein (34). These works provided the first evidence that changes in the primary structure of an Ag affecting speed of degradation can have strong consequences for efficient CP. However, that the primary structure of an Ag can affect CP and not DP while not involving rapid degradation has not been previously described.

Previous work in other laboratories has shown that altering the flanking sequences of some Ags may affect DP. For example, Shastri et al. (35) showed that specific residues immediately following the SIINFEKL can affect DP. In the same vein, Eisenlohr and co-workers demonstrated specific negative effects on DP of point mutations immediately preceding or following an influenza nucleoprotein and that deletion of an AA immediately following this determinant was deleterious in the DP of short but not long constructs (3638). In addition, Mo et al. (39) showed that the specific sequences of the 8 and 5 AA preceding and following SIINFEKL, respectively, can affect DP. All these results imply that altering the immediate flanking sequences of a determinant can affect their processing by the proteasome and other peptidases. Here, our focus has been different. While some of other constructs included changes in the specific AA sequence (most of which did not affect DP) we have mostly focused on changes in the length of the antigen protein and the differential effects on DP vs. CP. For this purpose, we have systematically altered the length of the N and C terminal extensions flanking the dominant Kb-restricted determinant of OVA to demonstrate that extensions of minimal length at both sides are essential for efficient CP but not for DP of SIINFEKL. Our work shows that CP of SIINFEKL does not occur when the determinant localizes exactly at the N or C terminus of the Ag and that extensions at both termini are essential for efficient CP. In the case of the N-terminal extension we did not determine the exact length requirement but clearly showed maximal CP for a construct with a 46 AA extension and poor CP when the extension was 18 AA long. In the case of the C-terminal extension we demonstrated the exact requirement for 22 AA because removal but not replacement of the C-terminal AA in 46-SIINFEKL-22 was sufficient to strongly decrease CP in a construct that, otherwise, is cross-presented with high efficiency. Remarkably, removal of this AA did not result in a protein with decreased stability, which is thought to play an important role in determining whether a protein is cross-presented or not.

While the length of the extensions were important for CP, the OVA specific sequences were not absolutely essential because even we do not know whether the cut-off length is exactly the same (and most likely is not), replacement of the natural OVA sequences at either termini for sequences of the same length but derived from GFP resulted in constructs with the same CP patterns as those with the original OVA sequence. However, substitutions of the natural 46 AA N-terminal extension in 46-SIINFEKL-121 with sequences from influenza virus NP [46(C-NP)-SIINFEKL-121] resulted in the loss of CP indicating that some particular sequences may be deleterious. The reason for this remains unknown.

With rapid degradation ruled out, the reason why SIINFEKL must have N and C terminal extensions of minimal length but not particular sequence is not clear. We have made numerous attempts to unveil the mechanism by appending commonly used tags (HA, poly-His) to constructs that are not cross-presented as a way to track them intracellulary by confocal microscopy or biochemically in co-immunoprecipitation experiments. Unfortunately, we did not reach any conclusive results because Ag tagging affected the CP of the constructs most likely because the tags alter the length and sequence of the constructs). Still, we speculate that size may affect the specific enzymes that participate in protein degradation, modification, or association with scaffolding proteins such as chaperones. Also, the extensions may themselves protect the determinant from proteolytic destruction. For example, an N-terminal extension could protect the amino terminus of the determinant from being rapidly chewed-off by amino peptidases. Nonetheless, our finding of differences in CP efficiency that cannot be explained by differential half-life of the Ag add an additional factor that may affect CP (i.e., length of the flanking AA chains) but do not contradict the work of Norbury et al. (17) showing that short antigenic half-life can decrease CP because we did not identify constructs with short-half lives that are nonetheless well cross-presented.

While most of our work was performed in tissue culture using bone marrow derived Mϕ as pAPC, we have used some of the constructs in vivo to show that the basic pattern of CP is maintained in an in vivo setting and without a predetermined pAPC. Clearly, 46-SIINFEKL-21 reproducibly induced less OT1 proliferation in vivo than 46-SIINFEKL-22. For example, the total number of OT1 cells (events, in Figure 6) in mice immunized with infected donor cells expressing 46-SIINFEKL-21 was decreased ~20% in the LN on day 4 and ~55% in the spleen on day 6 after immunization when compared with those immunized with infected donor cells expressing 46-SIINFEKL-22. The differences between these two constructs in vivo, however, were not as pronounced as in vitro (~80% reduction) but this may most likely be explained by the high sensitivity of the OT1 system and that measuring OT1 proliferation in vivo requires several days. Nevertheless, similar to the in vitro assay (not shown) CP of 46-SIINFEKL-16 was completely abolished in vivo. It is also important to note that in our in vitro and in vivo experiments the synthesis of the Ag is driven but not encoded by the infecting VACV and can only occur in the donor cells because they express the T7 polymerase (16). Thus, even though VACV can replicate in A9 cells and spread to other cells, our system allowed us to measure CP without having to inactivate the virus. Hence, in the in vitro experiments feeding Mϕ with infected cells resulted in their activation (not shown) while the in vivo experiments reflect CP as it occurs during the inflammatory conditions that are normally induced by live VACV infection and have been shown to utilize TLR2 (40, 41).

In this report we exclusively use variants of the model antigen OVA for which there are very well defined reagents that allow this type of study. Specific details of our findings should not be considered as rules since it is very likely that the exact overall length of the expressed polypeptide and that of the extensions are intrinsic to the OVA model and will not be identical for other antigens. Indeed, for some determinants, flanking sequences may not be required as recently shown for a Db restricted peptide from influenza virus (18). However, our studies show by clear example that the primary structure of the antigen may strongly affect the efficiency of CP and this possibility should be considered for vaccines that use CP as the main mechanism for TCD8+ stimulation such as DNA vaccines.

To date little is known about the relative contributions of CP and DP to anti-viral TCD8+ responses and whether this may vary for each type of virus. Our work demonstrates that CP can be much more limited by the primary structure of the Ag than DP and may explain why some Ags that are presented directly are not cross-presented. Our studies identified several OVA constructs with minimal differences in their sequence that are excellent substrates for DP but that vary widely as substrates for CP. Side-by-side comparison of these constructs expressed in recombinant viruses for their ability to initiate TCD8+ responses in vivo may provide direct evidence about the relative importance of CP and DP for the priming of anti-viral TCD8+.

Figure 8
The rules of in vitro CP and in vivo cross-priming are similar


We thank Dr. Bernard Moss for essential reagents, Drs. Glenn Rall, Laurence Eisenlohr and Daniel Rubio for critical reading of the manuscript, Ms. Holly Gillin for secretarial assistance, and Fox Chase Cancer Center Laboratory Animal, Sequencing and Flow Cytometry facilities for resources.


1This work was supported by NIH grants R01AI048849 and R21AI058179 to LJS and CA00697 to Fox Chase Cancer Center.


1. Townsend AR, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell. 1986;44:959–968. [PubMed]
2. Townsend A, Bodmer H. Antigen recognition by class I-restricted T lymphocytes. Annu Rev Immunol. 1989;7:601–624. [PubMed]
3. Townsend AR, Bastin J, Gould K, Brownlee GG. Cytotoxic T lymphocytes recognize influenza haemagglutinin that lacks a signal sequence. Nature. 1986;324:575–577. [PubMed]
4. Townsend AR, Gotch FM, Davey J. Cytotoxic T cells recognize fragments of the influenza nucleoprotein. Cell. 1985;42:457–467. [PubMed]
5. Sigal LJ, Crotty S, Andino R, Rock KL. Cytotoxic T-cell immunity to virus-infected non-haematopoietic cells requires presentation of exogenous antigen. Nature. 1999;398:77–80. [PubMed]
6. Sigal LJ, Rock KL. Bone marrow-derived antigen-presenting cells are required for the generation of cytotoxic T lymphocyte responses to viruses and use transporter associated with antigen presentation (TAP)-dependent and - independent pathways of antigen presentation. J Exp Med. 2000;192:1143–1150. [PMC free article] [PubMed]
7. Bevan MJ. Minor H antigens introduced on H-2 different stimulating cells cross-react at the cytotoxic T cell level during in vivo priming. J Immunol. 1976;117:2233–2238. [PubMed]
8. Bevan MJ. Cross-priming for a secondary cytotoxic response to minor H antigens with H-2 congenic cells which do not cross-react in the cytotoxic assay. J Exp Med. 1976;143:1283–1288. [PMC free article] [PubMed]
9. Norbury CC, Sigal LJ. Cross priming or direct priming: is that really the question? Curr Opin Immunol. 2003;15:82–88. [PubMed]
10. Rock KL. A new foreign policy: MHC class I molecules monitor the outside world. Immunology Today. 1996;17:131–137. [PubMed]
11. Yewdell JW, Norbury CC, Bennink JR. Mechanisms of exogenous antigen presentation by MHC class I molecules in vitro and in vivo: implications for generating CD8+ T cell responses to infectious agents, tumors, transplants, and vaccines. Adv Immunol. 1999;73:1–77. [PubMed]
12. Huang AY, Bruce AT, Pardoll DM, Levitsky HI. In vivo cross-priming of MHC class I-restricted antigens requires the TAP transporter. Immunity. 1996;4:349–355. [PubMed]
13. Cho JH, Youn JW, Sung YC. Cross-priming as a predominant mechanism for inducing CD8(+) T cell responses in gene gun DNA immunization. J Immunol. 2001;167:5549–5557. [PubMed]
14. Corr M, von Damm A, Lee DJ, Tighe H. In vivo priming by DNA injection occurs predominantly by antigen transfer. J Immunol. 1999;163:4721–4727. [PubMed]
15. Leifert JA, Rodriguez-Carreno MP, Rodriguez F, Whitton JL. Targeting plasmid-encoded proteins to the antigen presentation pathways. Immunol Rev. 2004;199:40–53. [PubMed]
16. Serna A, Ramirez MC, Soukhanova A, Sigal LJ. Cutting Edge: Efficient MHC Class I Cross-Presentation during Early Vaccinia Infection Requires the Transfer of Proteasomal Intermediates between Antigen Donor and Presenting Cells. J Immunol. 2003;171:5668–5672. [PubMed]
17. Norbury CC, Basta S, Donohue KB, Tscharke DC, Princiotta MF, Berglund P, Gibbs J, Bennink JR, Yewdell JW. CD8+ T cell cross-priming via transfer of proteasome substrates. Science. 2004;304:1318–1321. [PubMed]
18. Lev A, Takeda K, Zanker D, Maynard JC, Dimberu P, Waffarn E, Gibbs J, Netzer N, Princiotta MF, Neckers L, Picard D, Nicchitta CV, Chen W, Reiter Y, Bennink JR, Yewdell JW. The exception that reinforces the rule: crosspriming by cytosolic peptides that escape degradation. Immunity. 2008;28:787–798. [PMC free article] [PubMed]
19. Elroy-Stein O, Moss B. Cytoplasmic expression system based on constitutive synthesis of bacteriophage T7 RNA polymerase in mammalian cells. Proc Natl Acad Sci U S A. 1990;87:6743–6747. [PubMed]
20. Elroy-Stein O, Moss B. Chapter 16. Gene expression using the vaccinia virus/T7 RNA polymerase hybrid system. Curr Protoc Mol Biol. 2001 Unit16 19. [PubMed]
21. Karttunen J, Sanderson S, Shastri N. Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens. Proc Natl Acad Sci U S A. 1992;89:6020–6024. [PubMed]
22. Elroy-Stein O, Moss B, editors. Gene expression using the vaccinia virus/T7 RNA polymerase. New York: Wiley Interscience; 1992.
23. Blasco R, Moss B. Selection of recombinant vaccinia viruses on the basis of plaque formation. Gene. 1995;158:157–162. [PubMed]
24. Ramirez MC, Sigal LJ. Macrophages and Dendritic Cells Use the Cytosolic Pathway to Rapidly Cross-Present Antigen from Live, Vaccinia-Infected Cells. J Immunol. 2002;169:6733–6742. [PubMed]
25. Kurts C, Kosaka H, Carbone FR, Miller JF, Heath WR. Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8(+) T cells. J Exp Med. 1997;186:239–245. [PMC free article] [PubMed]
26. Lyons AB, Parish CR. Determination of lymphocyte division by flow cytometry. J Immunol Methods. 1994;171:131–137. [PubMed]
27. Craiu A, Akopian T, Goldberg A, Rock KL. Two distinct proteolytic processes in the generation of a major histocompatibility complex class I-presented peptide. Proc Natl Acad Sci U S A. 1997;94:10850–10855. [PubMed]
28. Rock KL, York IA, Goldberg AL. Post-proteasomal antigen processing for major histocompatibility complex class I presentation. Nat Immunol. 2004;5:670–677. [PubMed]
29. Hogquist KA, Jameson SC, Heath WR, Howard JL, Bevan MJ, Carbone FR. T cell receptor antagonist peptides induce positive selection. Cell. 1994;76:17–27. [PubMed]
30. Zinkernagel RM. On cross-priming of MHC class I-specific CTL: rule or exception? Eur J Immunol. 2002;32:2385–2392. [PubMed]
31. Melief CJ. Mini-review: Regulation of cytotoxic T lymphocyte responses by dendritic cells: peaceful coexistence of cross-priming and direct priming? Eur J Immunol. 2003;33:2645–2654. [PubMed]
32. Zinkernagel RM. Localization dose and time of antigens determine immune reactivity. Semin Immunol. 2000;12:163–171. discussion 257–344. [PubMed]
33. Ochsenbein AF, Klenerman P, Karrer U, Ludewig B, Pericin M, Hengartner H, Zinkernagel RM. Immune surveillance against a solid tumor fails because of immunological ignorance. Proc Natl Acad Sci U S A. 1999;96:2233–2238. [PubMed]
34. Wolkers MC, Brouwenstijn N, Bakker AH, Toebes M, Schumacher TN. Antigen bias in T cell cross-priming. Science. 2004;304:1314–1317. [PubMed]
35. Shastri N, Serwold T, Gonzalez F. Presentation of endogenous peptide/MHC class I complexes is profoundly influenced by specific C-terminal flanking residues. J Immunol. 1995;155:4339–4346. [PubMed]
36. Yellen-Shaw AJ, Wherry EJ, Dubois GC, Eisenlohr LC. Point mutation flanking a CTL epitope ablates in vitro and in vivo recognition of a full-length viral protein. J Immunol. 1997;158:3227–3234. [PubMed]
37. Yellen-Shaw AJ, Eisenlohr LC. Regulation of class I-restricted epitope processing by local or distal flanking sequence. J Immunol. 1997;158:1727–1733. [PubMed]
38. Eisenlohr LC, Yewdell JW, Bennink JR. Flanking sequences influence the presentation of an endogenously synthesized peptide to cytotoxic T lymphocytes. J Exp Med. 1992;175:481–487. [PMC free article] [PubMed]
39. Mo AX, van Lelyveld SF, Craiu A, Rock KL. Sequences that flank subdominant and cryptic epitopes influence the proteolytic generation of MHC class I-presented peptides. J Immunol. 2000;164:4003–4010. [PubMed]
40. Quigley M, Martinez J, Huang X, Yang Y. A critical role for direct TLR2-MyD88 signaling in CD8 T cell clonal expansion and memory formation following vaccinia viral infection. Blood. 2008 [PubMed]
41. Zhu J, Martinez J, Huang X, Yang Y. Innate immunity against vaccinia virus is mediated by TLR2 and requires TLR-independent production of IFN-beta. Blood. 2007;109:619–625. [PubMed]