Exogenous, TAP‐independent lysosomal presentation of a respiratory syncytial virus CTL epitope

Respiratory syncytial virus causes lower respiratory tract infections in infancy and old age, affecting also immunocompromised patients. The viral fusion protein is an important vaccine candidate eliciting antibody and cell‐mediated immune responses. CD8+ cytotoxic T lymphocytes (CTLs) are known to have a role in both lung pathology and viral clearance. In BALB/c mice, the fusion protein epitope F249‐258 is presented to CTLs by the murine major histocompatibility complex (MHC) class I molecule Kd. In cells infected with recombinant vaccinia viruses encoding the fusion protein, F249‐258 is presented by MHC class I molecules through pathways that are independent of the transporters associated with antigen processing (TAP). We have now found that F249‐258 can be generated from non‐infectious virus from an exogenous source. Antigen processing follows a lysosomal pathway that appears to require autophagy. As a practical consequence, inactivated virus suffices for in vivo priming of virus‐specific CTLs.


INTRODUCTION
CD8 þ cytotoxic T lymphocytes (CTLs) specifically recognise major histocompatibility complex (MHC) class I molecules presenting viral peptides on the surface of infected cells. In general, presented peptides are produced after cytosolic proteolytic processing by the proteasome of newly synthesised viral proteins. Cytosolic viral peptides are then efficiently translocated by the transporter associated with antigen processing (TAP) to the lumen of the endoplasmic reticulum, from where the MHC/peptide complexes reach the cell surface following the constitutive secretory route. Alternative pathways can present antigens independently of TAP by diverse mechanisms (reviewed in Johnstone and Del Val 1 and Del Val et al. 2 ). Peptides presented on MHC class I molecules are usually processed from antigens synthesised within the infected cell. However, professional antigenpresenting cells can process exogenous antigens and cross-present peptides to CTLs.
Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in infants and young children, 3 affecting also immunocompromised patients and the elderly. The fusion (F) glycoprotein of RSV is a major vaccine candidate as it is an important target for neutralising antibodies 4 and virus-specific CTLs. 5 Studies in the mouse model of human RSV have shown that CTLs have a role both in lung pathology and viral clearance. 6,7 In previous studies, CTL epitope F249-258 presented by the murine MHC class I molecule K d was identified in the RSV F protein, 8 and found to be presented only through TAP-independent pathways 9 in cells infected with RSV or recombinant vaccinia virus (rVACV). We now report on the further characterisation of TAP-independent presentation of epitope F249-258 from rVACV and reveal that epitope F249-258 is processed from exogenous precursors present in the virus inoculum. Exogenous antigen follows a TAP-independent lysosomal MHC class I antigenpresentation pathway that appears to require autophagy for CTL activation. Interestingly, CTLs are also activated by UV-inactivated (UVi) non-infectious rVACV in vivo.

RESULTS AND DISCUSSION
TAP-independent presentation of F249-258 follows an exogenous lysosomal pathway In a previous study, 9 we found that epitope F249-258 is presented by K d independently of TAP in cells infected with RSV or with rVACV expressing either native F (vvF) or even cytosolic Fsig À protein (vvFsig À ) lacking the signal peptide ( Figure 1a). In cells infected with rVACV, TAP-independent presentation of epitope F249-258 was partially resistant to brefeldin A, a metabolite that specifically blocks anterograde transport from the Golgi complex in the endogenous secretory pathway. 9 Presentation was also partially resistant to lactacystin, a proteasome inhibitor. 9 Such an atypical behaviour of epitope F249-258 suggested an exogenous processing pathway. To assess the requirement for infectious virus, rVACV vvF and vvFsig À infectious particles were inactivated by UV light. Absence of infectivity was controlled for the UVi rVACV by titration on CV-1 monolayers that revealed no p.f.u., and absence of protein expression from UVi rVACV was confirmed by flow cytometry in infected CV-1 and T2/K d cells (Figure 1b). Virus titration was always used as it is more sensitive to control inactivation of UVi rVACV, because CV-1 cells infected with vvF at m.o.i. 0.001 were not stained with F-specific antibody but, however, p.f.u. were observed. Of note, the full-length proteins were not detected by cytometry when UVi viruses adsorbed to cells (Figure 1b).
To detect viral antigen presentation, intracellular cytokine staining (ICS) assays were performed, in which TAP-deficient T2/K d cells served as targets to activate CTL F/F249-258. This monospecific CTL line was generated in vitro by stimulating splenocytes obtained from vvF-infected BALB/c mice with peptide F249-258. Figure 1c shows that CTLs were activated by T2/K d and dendritic cells, either infected with rVACV (light grey bars) or with the equivalent volume of the UVi counterparts (dark grey bars). These results suggest that TAPindependent presentation may be due to an exogenous source of F249-258 that was not synthesised endogenously in infected cells, but present in the purified virus inoculum. The equal behaviour of infectious and non-infectious rVACV suggests the existence of a sole exogenous processing pathway. In contrast, in cells expressing the native F protein from its natural context, RSV, we described TAPindependent endogenous presentation of F249-258. 9 Detection of CTL activation occurs above a threshold established by a certain amount of MHC/peptide complexes exposed on the surface of the infected cell. Thus, we cannot rule out the possibility that both the endogenous and exogenous TAP-independent F249-258 presentation pathways coexist in cells infected with rVACV, but we are currently unable to discriminate among them in our experimental setting. However, we are certain of addressing exclusively the exogenous pathway when UVi rVACV is used. Dependence on protein synthesis was studied by infecting T2/K d cells with vvF and vvFsig À or with their UVi counterparts in the presence of protein-synthesis inhibitor cycloheximide. Conventional presentation of viral proteins requires their endogenous expression in infected cells, and thus activation of CTLs is completely blocked by cycloheximide. In contrast, Figure 1d does not show a strict proteinsynthesis requirement for F249-258 presentation to CTLs. This result is compatible with the presence of epitope precursors in the virus inoculum that, in order to complete antigen processing, may require some protein synthesis of processing proteases or other cellular factors.
To evaluate if the peptide that represents the final epitope was already present in the virus inoculum, infection of T2/K d cells with vvF or vvF UVi was not allowed to proceed beyond viral adsorption (Figure 1e). Limiting concentrations of the synthetic peptide representing the final epitope were added in parallel to target cells immediately before fixing, and strong CTL activation was observed (x in Figure 1e). Absence of CTL activation at time 0 min after virus addition thus allows concluding that the final peptide itself was not present in the purified rVACV inoculum. Indeed, time was required to induce activation of CTLs, probably for the processing of precursors present in the virus inoculum.
TAP-independent processing followed by loading of exogenous antigen-derived peptides onto recycling MHC class I molecules has been shown to occur in endolysosomal acidic compartments of nonprofessional antigen-presenting cells. 10-12 MHC class II extracellular antigen processing by dendritic cells is also affected by macroautophagy. 13,14 The requirement found for further processing of precursors of epitope F249-258 may in fact point to a lysosomal or autophagy pathway. In general, autophagy supplies cytoplasmic components to lysosomes for their degradation. However, the F glycoprotein might reach lysosomes either by autophagy or as a final step after signal-peptide-mediated protein insertion into the endoplasmic reticulum. Therefore, to address the different lysosomal processing pathways, including autophagy, cells were infected with virus expressing cytosolic Fsig À , thus theoretically allowing antigen access to lysosomal compartments endogenously from the cytosol only through autophagy (vvFsig À ) or exogenously from the inoculum (vvFsig À and vvFsig À UVi). ICS assays were performed in the presence of NH 4 Cl, a lysosomotropic agent that raises intralysosomal pH, leupeptin (Leup), which inhibits trypsin-like and cysteine lysosomal proteases, 15 NH 4 Cl combined with Leup, which blocks different forms of autophagy, including chaperonemediated autophagy, as well as lysosomal degradation, and 3-methyladenine, 16 which blocks both endocytic and endogenous protein degradation through macroautophagy. 17 Figure 1f shows strong inhibitions of CTL activation by all the inhibitors tested except by Leup. In addition, similar results were obtained with chloroquine, another lysosomotropic agent (data not shown). In all cases, inhibitions seemed higher for UVi rVACV than for infectious rVACV. Therefore, the data suggest that precursors of epitope F249-258 followed a lysosomal pathway to be processed to the final epitope presented to CTLs. MHC class I presentation of an exogenous antigen has been shown to be carried out in these acidic compartments by the Leup-sensitive lysosomal protease cathepsin S. 18 Our results suggest that epitope F249-258 precursors from exogenous sources follow a proteasome-independent and TAP-independent lysosomal pathway that does not require Leup-sensitive endolysosomal serine or cysteine proteases and appears to involve autophagy for epitope presentation to CTLs. At this stage, it is unclear how exogenous precursors may reach lysosomes by a form of autophagy, but a role for the fusion activity of the F protein was unlikely, as the cytosolic Fsig À protein was equally presented to CTLs by this exogenous, TAP-independent lysosomal pathway. Alternatively, rather than for processing, intact lysosomes may be required to allow entry into the cytosol of infectious or UVi virion cores 19 if they carried epitope F249-258 precursors, or to allow entry of such precursors if they used the same route as the virions. Finally, exogenous precursors may employ some of the autophagy machinery recruited to the plasma membrane as described for bacterial infection. 20 Lysosomes 12 and, more recently, autophagy have been reported to participate also in endogenous MHC class I presentation of viral epitopes through a proteasome-and TAP-dependent pathway 21 and a proteasome-and TAP-independent pathway. 22 Autophagy also supplies MHC class II molecules with peptides for presentation of cytosolic antigens. 23 In particular, presentation of epitope F249-258 from cells expressing the cytosolic Fsig À protein is very suggestive of an additional TAP-independent endogenous processing of this F protein by autophagy. No evidence was found for an involvement of chaperone-mediated autophagy, as the inhibitory effect of NH 4 Cl combined with Leup was not higher than that of 3-methyladenine ( Figure 1f). However, the presence of the exogenous pathway, revealed by testing UVi rVACV, precludes the detailed study of additional endogenous pathways and their contribution to overall TAP-independent presentation of these proteins.

Cross-priming of CD8 þ T lymphocytes specific for F249-258 in vivo
To assess in vivo the relevance of the exogenous lysosomal presentation pathway described in cell culture for epitope F249-258, mice were vaccinated with infectious vvF and non-infectious UVi vvF. The acute CD8 þ T-lymphocyte response was studied 5 or 6 days after BALB/c mice immunisation by performing ex vivo ICS assays with cells obtained from the spleen or peritoneal exudate. vvF (light grey bars) primed CD8 þ T lymphocytes specific for F249-258 at levels previously described. 8 Non-infectious UVi vvF (dark grey bars) activated CD8 þ T lymphocytes at lower levels, decreasing with respect to vvF eight to three times in spleen and peritoneal exudate, respectively (Figure 2a), supporting the hypothesis of coexisting endogenous and exogenous TAP-independent F249-258 presentation pathways in cells infected with vvF. Interestingly, when splenocytes from UVi vvF-immunised mice were cultured, stable CTL lines were generated, and they not only recognised peptide-pulsed cells (data not shown) but also cells infected with vvFsig À (Figure 2b). Therefore, F249-258-specific CTLs are cross-primed in vivo by non-infectious virus, presumably by the exogenous proteasome-independent TAPindependent lysosomal pathway. Although we do not know the exact nature of the antigenic precursor source of epitope F249-258, it must include a portion of the F protein harbouring the epitope, which is interestingly located near important antigenic sites for neutralising antibodies. 24 Our findings have important implications for RSV vaccine studies as they open the possibility of priming RSV-specific CTLs and antibodies with a non-infectious agent.

METHODS Mice
BALB/c mice (H-2 d haplotype) (The Jackson Laboratory, Bar Harbor, ME, USA) were bred in our colony in accordance to national regulations. Animal studies have been reviewed and approved by the appropriate institutional review committee. Mice were infected intraperitoneally with 10 7 p.f.u. of vvF or with an equal volume of UVi vvF containing o10 p.f.u. after inactivation.
CTL priming by non-infectious virus C Johnstone et al rVACVs Construction of rVACV vvF containing the F gene of the Long strain of RSV has been described. 25 Recombinant vvFsig À encodes a cytosolic F protein in which the signal peptide has been deleted. 26 As a result, the protein is rapidly degraded and is barely detectable in infected cells. All viruses were based on the Western Reserve wild-type vaccinia strain. Virus stocks were purified by ultracentrifugation of crude infected cell lysates through a 36% sucrose cushion. Inactivation of rVACV was performed by exposing rVACV to 254 nm UV light for 30-60 min at 4 1C. Inactivation of the UVi rVACV was controlled by viral titration on CV-1 cell monolayers to confirm absence of p.f.u. after 24 h infection. Lack of protein expression was monitored by flow cytometry staining with anti-RSV F protein monoclonal antibody (clone 18B2, Argene, Verniolle, France). Infectious titer was reduced below detection limit by at least 10 6 fold.

Cell lines and virus infection
T2/K d is a human lymphoblastoid T2 cell line deficient in TAP and transfected with K d , and was provided by Dr G Hämmerling (German Cancer Research Centre, Heidelberg, Germany). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 5 Â 10 À5 M 2-ME in a 5% CO 2 atmosphere at 37 1C. CV-1 cells are green monkey kidney fibroblasts and were grown in Dulbecco's Modified Eagle medium. Bone marrow-derived BALB/c dendritic cells were obtained as described. 27 Infection of cells with rVACV was performed at a m.o.i. of 3-15 as described. 9,28 Protein synthesis inhibitor cycloheximide (Sigma, St Louis, MO, USA) was used at 50 mg ml À1 during infection. NH 4 Cl was prepared fresh and used at 20 mM. Leup (Sigma) was used at 100 mM. 3-methyladenine (Sigma) was prepared fresh in medium and used at 10 mM.

CTL lines and detection of CD8 þ T-lymphocyte activation by ICS
Generation of polyclonal CTL line CTL F/F249-258 has been described. 8 Briefly, splenocytes were obtained from vvF-infected or UVi vvF-inoculated BALB/c mice and CTL lines generated by in vitro stimulation with 10 À9 M peptide F249-258 (TYMLTNSELL).
ICS assays to measure CTL activation were performed as described. 9 Target cells were fixed in 1% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) at 4 1C for 10 min and washed twice before being used to activate CTLs by coculture. In ex vivo ICS, splenocytes or peritoneal exudate cells were obtained from BALB/c mice 5 or 6 days post infection with 10 7 p.f.u. vvF or UVi vvF, stimulated with an excess 10 À7 M peptide for up to 2 h, and further stimulated for 3 h in the presence of brefeldin A. Following stimulation during coculture, cells were stained and events acquired and analysed as described. 9 BALB/c mice a b  Figure 2 CD8 þ T lymphocytes specific for epitope F249-258 are crossprimed in vivo by non-infectious UVi vvF. (a) Ex vivo ICS assays were performed with splenocytes or peritoneal exudate cells (PEC) obtained from BALB/c mice 5 or 6 days after immunisation with 10 7 p.f.u. vvF (light grey bars) or UVi vvF (dark grey bars). Percentage of total CD8 þ lymphocytes that were stained with anti-IFNg after activation with peptide F249-258 is represented. The mean of two independent experiments with a total of two and five mice infected with vvF or UVi vvF, respectively, is represented and the s.e. of the mean indicated by error bars. (b) Splenocytes obtained from one of the five BALB/c mice inoculated with UVi vvF were stimulated in vitro to generate a CTL F/F249-258 line. An ICS assay was performed with the obtained CTL line and T2/K d cells infected with the indicated virus. Percentage of total CD8 þ lymphocytes that expressed IFNg is represented. F249-258 peptide-coated cells activated 60% of the CTL line CD8 þ T lymphocytes.