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Inhibitory Effects of Cytomegalovirus Proteins US2 and US11 Point to Contributions from Direct Priming and Cross-Priming in Induction of Vaccinia Virus-Specific CD8⁺ T Cells

Sameh Basta^*, Weisan Chen, Jack R. Bennink^* and Jonathan W. Yewdell^1,*

^* Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892; and Cancer Vaccine Unit, Ludwig Institute for Cancer Research, Austin and Repatriation Medical Center, Heidelberg, Victoria, Australia

	Abstract

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The extent to which naive CD8⁺ CTLs (T_CD8⁺) are primed by APCs presenting endogenous Ags (direct priming) or Ags acquired from other infected cells (cross-priming) is a critical topic in basic and applied immunology. To examine the contribution of direct priming in the induction of VV-specific T_CD8⁺, we generated recombinant vaccinia viruses that express human CMV proteins (US2 and US11) that induce the destruction of newly synthesized MHC class I molecules. Expression of US2 or US11 was associated with a 24–63% decrease in numbers of primary or secondary VV-specific T_CD8⁺ responding to i.p. infection. Using HPLC-isolated peptides from VV-infected cells, we show that US2 and US11 selectively inhibit T_CD8⁺ responses to a subset of immunogenic VV determinants. Moreover, VV-US2 and lysates from VV-infected histoincompatible cells elicit T_CD8⁺ specific for a similar subset of VV determinants. These findings indicate that US2 and US11 can function in vivo to interfere with the activation of virus-specific T_CD8⁺. Furthermore, they suggest that 1) both cross-priming and direct priming contribute significantly to the generation of VV-specific T_CD8⁺, 2) the sets of immunogenic vaccinia virus determinants generated by cross-priming and direct priming are not completely overlapping, and 3) cross-priming overrides the effects of cis-acting viral interference with the class I Ag presentation pathway.

	Introduction

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CD8⁺ CTLs (T_CD8⁺)² play an important role in the immune responses to many viruses (1). The mechanisms by which T_CD8⁺ are elicited in vivo are just now being elucidated. It is thought that naive antiviral T_CD8⁺ are exclusively activated by professional APCs (pAPCs) (2, 3), which are the only cells in an organism that express the appropriate costimulatory molecules. Of particular interest is the source of peptides that find their way to the grooves of MHC class I molecules on the surface of pAPCs. In general terms, there are two potential sources of Ags: viral proteins that are synthesized by the APC and those that are obtained from other infected cells. The former mechanism of priming is known as direct priming and the latter is referred to as cross-priming (4).

These two priming mechanisms are not mutually exclusive. Rather, it is likely that both can contribute to priming depending on the exact conditions used. Variables include the nature of the virus and dose administered, the route of immunization, and the properties of the determinant and source protein (5, 6). Knowledge of the priming mechanisms is important for improving the ability of recombinant viruses to elicit T_CD8⁺ responses. One of the more important viruses in this regard is vaccinia virus (VV), which is being used in numerous clinical trials for eliciting T_CD8⁺ responses, in addition to its roles in eradicating the small pox agent variola virus and serving as an important vector in mouse model systems.

To examine the role of direct priming in the anti-VV T_CD8⁺ response, we inserted into VV genes encoding human CMV (HCMV) proteins US2 or US11 glycoproteins. These proteins are capable of destroying newly synthesized class I molecules by directing them to the cytosol where they are degraded by proteasomes (7). The ability of US2 or US11 to affect T_CD8⁺ responses in vivo has not been previously addressed. Because their actions ought to be limited strictly to virus-infected cells, any deleterious effects they exert on induction of T_CD8⁺ can be attributed to the contribution of direct priming. Using VV encoding US2 or US11 we show that both direct priming and cross-priming play important roles in the activation of local and systemic VV-specific T_CD8⁺ following i.p. infection.

	Materials and Methods

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Mice and cell lines

Female C57BL/6 (B6) mice and B6 I-A^b -chain^-/- (class II^-/-) (8) and D^boK^bo (9) mice were obtained from Taconic Farms (Germantown, NY). The mastocytoma cell line P815 (H-2^d) and the dendritic cell (DC) line DC2.4 (H-2^b) (10) were maintained in RPMI 1640 containing 10% FBS. 143b human osteosarcoma cells were maintained in DMEM containing 10% FBS. All media were purchased from Invitrogen (Gaithersburg, MD).

Recombinant VVs

rVVs encoding K^b, D^b, US2, US11 under the control of the p7.5 promoter, and -galactosidase under the control of the p11 promoter were generated by homologous recombination into the viral thymidine kinase gene as described (11). Virus only-expressing -galactosidase under the control of the p11 promoter was used as a control virus (VV-Con). Cells were infected with rVV (10 PFU/cell) in 200 µl of balanced salt solution (BSS) supplemented with 0.1% (w/v) BSA (BSS/BSA) at 37°C. After 1 h, 1 ml of DMEM containing 10% FCS was added for an additional 4 h, and the cells then washed and stained for cytofluorography. For in vivo infections, mice were injected i.p. with 10⁷ PFU of each rVV per mouse. In certain experiments ovaries were harvested at different time points and VV was titrated by plaquing on 143b cells.

Flow cytometry

For the detection of MHC class I expression, cells were washed in cold BSS/BSA and then stained with H-2K^b (AF6.88.5) and H-2D^b (KH95) using PE-conjugated mAbs (BD PharMingen, San Diego, CA). mAb binding was quantitated cytofluorographically after two washing cycles. For intracellular cytokine staining (ICS), cells were stained as previously described (12). Briefly, splenocytes or peritoneal lavages were resuspended in RPMI 10 at 2 x 10⁷/ml, and 100 µl were added per well to round-bottom 96-well plates. Infected cells (2 x 10⁵) were added to the wells for 2 h at 37°C, before the addition of brefeldin A (10 µg/ml) for another 3 h. Cells were then incubated for 20 min on ice with Cy5-conjugated mouse anti-CD8. Following fixation with 1% paraformaldehyde in PBS at room temperature for 20 min, the cells were incubated overnight with fluorescein-conjugated mouse anti-IFN- (BD PharMingen) and/or PE-conjugated TNF- diluted 1/150 in PBS containing 0.1% (w/v) saponin. Cells were acquired with the use of FACSCalibur (BD Biosciences, Mountain View, CA) and analyzed by the FLOWJO software (Tree Star, San Carlos, CA).

Extraction of cellular peptides and fractionation by reversed-phase HPLC

Peptides complexed to the H-2^b haplotype after VV infections were recovered and analyzed as previously described (13). In brief, cultures of DC2.4 cells were expanded in roller bottles. Cells (1 x 10⁹) were infected with VV as described above and incubated for an additional 6 h at 37°C before being pelleted. The pellets were washed in PBS, lysed, and further disrupted using a TenBroek tissue homogenizer (Wheaton Science Products, Millville, NJ). Lysates were sonicated and centrifuged at 10,000 x g for 30 min, and the supernatants were passed through a 3K cutoff filter (Macrosep filtron 3K; Pall Filtron, Northborough, MA). Samples (500 µl) were fractionated on a C18 column (Deltapack; Waters, Milford, MA) at 1 ml/min on trifluoroacetic acid/acetonitrile gradient. For the peptide binding assays 2 µl were used from the HPLC-collected fractions (50 µl).

Preparation of VV-infected HeLa cell lysates

HeLa cells were infected with VV at 10 PFU/cell for 12 h, gamma-irradiated (20,000 rad), and washed. Cells were resuspended at a concentration of 1.5 x 10⁷/ml in BSS/BSA and UV-irradiated on ice for 20 min at 2.7 mW/cm² using the UV Stratalinker 1800 (Sratagene, La Jolla, CA). Cells were washed twice and freeze-thawed three times. These steps were undertaken to avoid transferring infectious VV with the cells. Lysates were titrated by plaquing on 143b cells as above and no plaques were detected (<2 PFU/ml). Mice were injected i.p. with the HeLa lysates (1.5 x 10⁶ cell equivalents per mouse), and responder cells were harvested 6 days later.

	Results

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Effect of HCMV glycoproteins on the expression of K^b and D^b

It was previously shown that rVVs encoding US2 and US11 induce the degradation of human class I molecules (14). To extend these findings to mouse class I molecules, we coinfected P815 cells (H-2^d) with rVVs expressing K^b or D^b and rVVs expressing US2, US11, or a control rVV (VV-Con). Class I cell surface expression was determined by binding of directly conjugated K^b- or D^b-specific mAbs as measured by flow cytometry.

As seen in Fig. 1, relative to coinfection with VV-Con, K^b expression was reduced 30% by US2 and 61% by US11, while D^b expression was reduced by 75% by either US2 or US11. These findings are in agreement with the previous biochemical analysis of Machold et al. (15) of the effects of US2 and US11 on K^b and D^b with one exception: in contrast to our findings, Machold et al. (15) failed to detect an effect of US2 on K^b. There are a number of possible reasons for this discrepancy, including 1) lower sensitivity of the biochemical method, 2) delayed action of US2 on K^b relative to its effects of D^b and human class I molecules, such that the effect was missed in the 20-min chase period used, and 3) differences in the systems (different cells, VV-encoded US2 vs transfected US2).

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Effects of US2 and US11 on MHC expression after rVV infection in vitro. P815 cells were infected with rVV expressing either Kb (a) or Db (b) at a multiplicity of infection of 10 PFU/cell for 40 min. Cells were then superinfected at a multiplicity of infection of 10 PFU/cell with the rVV indicated. After 4 h, class I cell surface expression was determined by flow cytometry. Data are corrected for background staining of Abs against VV-infected cells lacking the corresponding class I molecule. Mean fluorescence values are shown at the top of each bar.

We next examined the immunogenicity of VV-US2 and VV-US11 relative to VV-Con. Mice were infected by i.p. injection with equivalent PFU of VV, and local and systemic T_CD8⁺ responses, respectively, were determined 6 days later by ICS of T_CD8⁺ recovered from peritoneal exudates and spleens. We measured the numbers of IFN-- and TNF--secreting T_CD8⁺ after ex vivo stimulation with P815 cells infected with VV-D^b or VV-K^b to assess VV-specific responses in association with each allomorph. Data are expressed as the percentage of VV-specific T_CD8⁺ relative to total CD8⁺ cells. Because similar numbers of CD8⁺ cells were recovered from mice immunized with the different rVVs, these data are representative of absolute numbers of responding T_CD8⁺. Roughly 60–80% of IFN--secreting cells also secrete TNF-. Because 95% of TNF--secreting cells also secrete IFN-, we will refer to these cells as IFN-/TNF--secreting cells. Subtracting the percentage of these cells from the total IFN--secreting cells reveals the number of cells exclusively secreting IFN-.

As seen in Fig. 2, a and b, we detected both K^b- and D^b-specific activation of VV-specific T_CD8⁺ secreting IFN-. As we observed previously following i.p. infection with influenza virus (12), a higher percentage of VV-specific T_CD8⁺ were present in peritoneal exudate cells (PECs) than in the spleen. Expression of US2 or US11 by VV was associated with a decrease in the numbers of VV-specific T_CD8⁺ (the percentage of decline in T_CD8⁺ is shown in Fig. 2, a and b, at the top of each column). The magnitude of the reduction ranged between 25 and 50%; similar effects were seen on IFN-/TNF--secreting T_CD8⁺. In contrast to their effects on class I expression in cultured cells (Fig. 1), US2 was actually slightly (but consistently) more suppressive than US11. In additional experiments, we found that US2 and US11 also inhibit VV-specific primary T_CD8⁺ responses in BALB/c mice (data not shown) and interfere with the generation of splenic memory T_CD8⁺ in B6 mice as assessed directly ex vivo (Fig. 2c).

View larger version (39K): [in this window] [in a new window]   FIGURE 2. Suppression of VV-specific TCD8+ responses by US2 and US11 in B6 mice. Splenocytes and PEC from B6 mice (a and b) were processed 6 days after i.p. infection with the VV indicated (VV-Con, open bars; VV-US2, hatched bars; or VV-US11, filled bars). c, Splenocytes were obtained from mice 30 days after infection. VV-specific TCD8+ were quantitated by ICS following in vitro stimulation with P815 cells infected with VV-Kb or VV-Db. All IFN--expressing cells are shown in left panels; right panels show IFN--positive cells that also express TNF-. Background values obtained by stimulation with uninfected P815 cells were subtracted from each panel; data represent the mean values from four individuals (SE is represented by the error bars). The numbers above the bars show the percentage of the response with VV-Con obtained following infection with US2 or US11.

View larger version (26K): [in this window] [in a new window]   FIGURE 3. rVVs replicate equally in vivo. a, The amounts of virus recovered from ovaries of B6 mice on the indicated day following i.p. infection was determined by plaque titration. b, Amount of virus recovered from KboDbo mice on day 6 postinfection. Data are expressed as mean ± SD from four mice per time point. For clarity, error bars are presented only for the VV-Con in a; errors on other samples are within these limits.

Contribution of T_CD4⁺ to US2- and US11-mediated interference with VV-specific T_CD8⁺ induction

US2 has been reported to induce the degradation of human MHC class II molecules and the class II accessory molecule DM (17). To examine the potential contribution of US2- or US11-mediated interference with class II molecules on their inhibition of VV-specific T_CD8⁺ responses, we compared the immunogenicity of VV-US2, VV-US11, and VV-Con in class II^-/- mice (Fig. 4). Consistent with previous findings (18), following infection with VV-Con, splenic VV-specific T_CD8⁺ responses were markedly decreased relative to responses in B6 mice. In absolute terms, numbers of responding T_CD8⁺ were decreased 10-fold, reflecting a 2.7-fold decrease in the overall T_CD8⁺ population and a 3.6-fold decrease in the fraction of Ag-specific T_CD8⁺. By contrast, there was only a slight decrease in peritoneal VV-specific T_CD8⁺.

View larger version (37K): [in this window] [in a new window]   FIGURE 4. Suppression of VV-specific TCD8+ responses by US2 and US11 in class II-/- mice. Spleen cells (a) and peritoneal cells (b) from MHC class II-/- mice were processed 6 days after i.p. infection with the VV indicated (VV-Con, open bars; VV-US2, hatched bars; or VV-US11, filled bars). VV-specific TCD8+ were quantitated by ICS following in vitro stimulation with P815 cells infected with VV-Kb or VV-Db. All IFN--expressing cells are shown in the left panels; right panels show IFN--positive cells that also express TNF-. Background values obtained by stimulation with uninfected P815 cells were subtracted from each panel; data represent the mean values from four individuals (SE is represented by the error bars). The numbers above the bars show the percentage of the response with VV-Con obtained following infection with US2 or US11.

These findings indicate that US2- and US11-mediated inhibition of VV-specific T_CD8⁺ induction occurs independently of any effects these proteins might have on MHC class II molecules.

Selective effects of US2 and US11 on VV-specific T_CD8⁺ specificity

The partial effects of US2 and US11 on induction of VV-specific T_CD8⁺ could reflect an incomplete effect on all specificities or more selective effects on a subset of specificities, or both of these factors. Due in part to the complexity of VV (200 or more gene products), viral peptides recognized by T_CD8⁺ have yet to be defined. Nevertheless, it is possible to dissect T_CD8⁺ responses into individual specificities (or at least subgroups of specificities) by HPLC fractionation of antigenic peptides recovered from virus-infected cells expressing the proper class I allomorphs.

As seen in Fig. 5a, fractionation of low-m.w. material present in lysates from VV-infected DC2.4 cells reveals at least 11 distinct antigenic activities that are able to activate d7 primary T_CD8⁺ ex vivo. This is likely to be a minimal estimate because, based on our experience with fractionating defined influenza virus peptides (19), there is a greater chance of multiple unrelated peptides eluting in the same fraction than there is of related peptides eluting in multiple fractions.

View larger version (40K): [in this window] [in a new window]   FIGURE 5. US2 and US11 interfere with VV-specific TCD8+ in a determinant-specific manner. DC2.4 (H-2b) cells were infected for 12 h, and the low-m.w. acid-soluble material was fractionated by reversed-phase HPLC. Two microliters of each fraction was then added to low temperature-induced DC2.4 cells at least 1 h before addition of PEC pooled from four B6 mice immunized 6 days earlier with VV-Con ( and blue line), VV-US2 ( and green line), VV-US11 ( and red line), or with VV-infected gamma-irradiated (20,000 rad) HeLa cells (1.5 x 106/mouse; and purple line) that were freeze-thawed three times and UV-irradiated to inactivate viral genomes associated with cells. The percentage of CD8+ cells responding to each fraction was determined by ICS. In b alternate fractions were tested for antigenicity. The percentage of acetonitrile is plotted as a dotted line on the Y2 axis.

Comparison of determinants primed by VV-US2 and infected histoincompatible cells

The data presented demonstrate that US2 and US11 interfere with induction of T_CD8⁺ specific for some VV-derived peptides, sparing responses to others. Because US2 and US11 should only interfere with direct priming, one potential explanation for this finding is that the peptides most sensitive to the effects of US2 and US11 are those that are exclusively or predominantly derived from direct priming. To test this idea, we compared the specificities of VV-specific T_CD8⁺ from immunized with VV-Con, VV-US2 (VV-US2 was chosen because it exhibits greater effects on the responding T_CD8⁺ repertoire), and lysates from VV-infected HeLa cells. By definition, HeLa cell-derived Ags are presented exclusively via cross-priming (we took care to inactivate residual infectious VV by UV irradiation).

PEC were obtained 6 days after immunization, and specificities of responding T_CD8⁺ were determined by ICS using HPLC fractions from VV-infected DC2.4 cells. This revealed a remarkably close concordance between the specificities elicited by US2 and those elicited by HeLa cells extracts (Fig. 5b). Although the correlation is not perfect, this is not surprising because 1) US2 is not expected to completely block direct priming, and 2) there are likely to be differences between cross-priming by injection of cell lysates and the natural route associated with VV infection. These findings support the idea that the partial effects of US2 on T_CD8⁺ induction are due to blocking the presentation of a subset of determinants that are uniquely generated by direct priming.

	Discussion

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We have shown that the HCMV proteins US2 and US11 interfere with the induction of T_CD8⁺ responses in B6 mice. These data, in conjunction with those of Siliciano et al.,³ provide the initial confirmation of the proposed in vivo function of these proteins, extending the finding that US11 interferes with Ag presentation in vitro (20). This provides an important clue as to the evolutionary significance of these proteins, but it is important to recognize that this evidence is not definitive. It is risky business to study the functions of proteins outside of their evolutionary context, and it is possible that US2 and US11 have additional functions that may be of equal or greater importance than destruction of MHC class Ia molecules.

In a previous study, we failed to detect any effect of expressing the adenovirus E19 glycoprotein on the induction of VV-specific T_CD8⁺ (21). One of the explanations we offered for this failure is that cross-priming is sufficiently robust to prevent detection of any effects E19 might have on direct priming. Our present findings strongly suggest that this is not the case, and it is interesting to note that the effects of VV-expressed E19 on MHC class I expression in cultured cells (22) are at least of equal magnitude to those exerted by either US2 or US11. Similarly, US2 was more effective than US11 at blocking K^b-restricted responses despite being less effective at blocking K^b expression in vitro. There are several potential explanations for these discrepancies.

First, the effects of viral interfering molecules on class I molecules may vary between cells used for in vitro studies and APCs that function in vivo. For example, US2 may be more effective at destroying K^b generated by APCs in vivo than VV-K^b-infected cells due to VV-induced or cell-specific differences in K^b structure or trafficking. Infected cell protein 47 has been reported to inhibit mouse T_CD8⁺ responses in vivo (23), although it has no detectable effects on Ag presentation in mouse cells when expressed by VV (our unpublished results). Cytokines (e.g., IFN-) that are released in vivo by NK cells and other innate effector cells responding to VV infection may differentially influence US2- and US11-mediated effects on class I molecules (24).

Second, US2 (or US11, for that matter) may affect other molecules that play a role in T_CD8⁺ priming. For example, US2 has been reported to destroy human class II molecules (17), although clearly this cannot be responsible for its effects on priming, which increase in class II^-/- mice. US2 also destroys the nonclassical class I molecule HFE, which is distantly related to classical class I molecules by sequence homology (25). E19, which was the first viral molecule shown to interfere with class I maturation (26, 27), is now known to interact with completely unrelated molecules. E19 was unexpectedly found to interact with TAP and interfere with its function (28), and we have observed that E19 inhibits the cell surface expression of mouse (but not human) ICAM1 (our unpublished observations). These examples provide graphic illustration that the interaction of viral immunomodulatory proteins with host proteins may be more degenerate than is generally considered. US2 and US11 may target multiple proteins involved in Ag presentation or costimulation on VV-infected DCs, which are likely to mediate direct priming in vivo (29). Finally, the ability of viral interfering molecules to block priming in vivo may depend on the precise kinetics of class I hindrance and the generation of immunogenic peptides.

A major goal of this study was to use US2 and US11 to examine the contributions of direct priming and cross-priming of T_CD8⁺ in VV-specific responses. Our findings support an important role for direct priming, extending a series of prior observations. Coupar et al. (30) observed that expression of proteins by early VV promoters often elicits stronger T_CD8⁺ responses than expression by late promoters, despite the latter resulting in the synthesis of greater amounts of protein in vivo, which presumably should function just as well for cross-priming. Bronte et al. (31) then showed that late VV gene expression in DCs is meager, which explains the findings of Coupar et al. (30) if infected DC are the principal priming APC.

The effects of US2 and US11 were magnified in class II^-/- mice. This is possibly due to a decrease in cross-priming in these mice, because in some circumstances cross-priming appears to be highly dependent on T_CD4⁺-mediated help (32). By this reckoning, the discrepancy we observe between the effects of US2 and US11 on T_CD8⁺ induction in splenic and peritoneal T_CD8⁺ in class II^-/- mice could mean that cross-priming of the residual splenic T_CD8⁺ is less dependent on T_CD4⁺-mediated help. There is a precedent for local and splenic T_CD8⁺ exhibiting distinct properties in studies of immunodominance hierarchies, where differences in the specificities of splenic and locally responsive antiviral T_CD8⁺ have been noted (33). In contrast, the requirement for T_CD4⁺ in cross-priming is not absolute. It has been reported that the T_CD4⁺ requirement for exogenous Ag presentation is inversely related to the dose of Ag used for immunization (34). Presumably, this reflects a decreased requirement for help with increasing numbers of peptide-class I complexes on APCs. Furthermore, robust cross-priming to tumor Ags was recently demonstrated in class II^-/- mice (35). Thus, we cannot eliminate the possibility that the increased effectiveness of US2 and US11 in these mice is due to general decrease in the sensitivity of responding T_CD8⁺, magnifying a partial reduction in peptide MHC complexes mediated by US2 and US11.

Perhaps our most interesting finding is that US2 and US11 selectively inhibit the generation of T_CD8⁺ to a subset of VV peptides that are poorly immunogenic when mice are immunized with VV-infected HeLa cell lysates. We believe that this subset represents determinants that are efficiently generated by infected pAPCs but inefficiently generated by cross-priming (A in Fig. 6). To our knowledge, this is the first description of such determinants. We also provide evidence that, as expected, there is considerable overlap in the determinants generated by the two priming pathways (B in Fig. 6). There are also likely to be some determinants that are uniquely immunogenic through cross-priming (C in Fig. 6), as has been demonstrated for other Ags (36, 37). Although the DC2.4 cells we used as source of VV peptides are known to mediate cross-presentation (10), these determinants may yet be invisible to our analysis due to inefficient presentation of exogenous Ags under the conditions used. Presumably, such determinants are relatively infrequent, because their cognate T_CD8⁺ would fail to recognize infected cells and would be limited to spewing their cytokines onto cross-presenting cells; however, this would be a neat way for T_CD8⁺ to specifically interact with cross-priming APCs. Additional experiments using cross-presenting cells are needed to identify T_CD8⁺ specific for such VV determinants and to examine their function in vivo.

View larger version (64K): [in this window] [in a new window]   FIGURE 6. Venn diagram of determinants generated by Ag processing pathways. In theory, direct priming and cross-priming should generate partially overlapping sets of peptides. The diagram shows priming mediated by virus-infected pAPC (left), which is blocked by US2/11 expressed by the virus. On the right is a non-APC peritoneal cell that provides viral Ags via the cross-priming pathway. US2/11 exclusively blocks the generation of peptides in set A, leaving those in set B and set C. Set C remains to be demonstrated experimentally.

Acknowlegements

We thank Beth Buschling for excellent technical assistance. We are grateful to Dr. Francois Lemonnier (Institut Pasteur, Paris, France), James Forman (University of Texas Southwestern, Dallas, TX) for providing D^boK^bo mice, and Dr. D. Tscharke for critical discussions.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Jonathan W. Yewdell, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Room 211, Building 4, 4 Center Drive, Bethesda, MD 20892-0440. E-mail address: jyewdell{at}nih.gov

² Abbreviations used in this paper: T_CD8⁺, CD8⁺ CTL; pAPC, professional APC; VV, vaccinia virus; PEC, peritoneal exudate cell; ICS, intracellular cytokine staining; DC, dendritic cell; HCMV, human CMV; BSS, balanced salt solution.

³ X. Shen, C. Buck, J. Wong, J. Zhang, and R. Siliciano. Direct priming and cross-priming contribute differentially to the induction of CD8⁺ CTL following exposure to vaccinia virus via different routes. Submitted for publication.

Received for publication November 26, 2001. Accepted for publication February 22, 2002.

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