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* Department of Microbiology and Immunology, University of Melbourne, Parkville, Australia;
Institute of Virology, University of Zurich, Zurich, Switzerland; and
Immunology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Australia
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The survival and life span of professional APC in vivo during the course of an immune response is relatively poorly understood. A couple of recent reports have demonstrated that Ag presentation is prolonged after HSV and influenza virus infection (5, 6). Although it is known that CTL can kill peptide-labeled dendritic cells (DC) in vivo (7, 8, 9, 10), little is known about the mechanisms involved in regulating these interactions. Medema et al. (11) demonstrated the up-regulation of a specific granzyme B inhibitor by DC following maturation by CD4+ T cells and maturation signals such as LPS and anti-CD40. This was shown to protect DC from CTL-mediated lysis in vitro.
In this study, we sought to examine the survival APC in vivo following infection with HSV. Ag presentation persists in the draining popliteal LN (PLN) in the presence of significant numbers of CTL (5). By transferring CTL specific for the immunodominant epitope from HSV glycoprotein B (gB498–505), we show the in vivo elimination of exogenous resting peptide-labeled DC by specific CTL, while maturation of these DC with LPS or anti-CD40 Ab prolonged their survival. Furthermore, pre-existing CTL abrogated endogenous Ag presentation within the draining PLN after HSV infection. However, cotransfer of activated virus-specific CD4+ T cells with the CTL prolonged Ag presentation in the PLN. Together these data suggest that signals that activate APC may be capable of preventing CTL-induced elimination of APC, thereby promoting prolonged Ag presentation in secondary lymphoid tissues.
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Materials and Methods |
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C57BL/6, gBT-I.1 (gBT-I) (12), OT-I (13), and OT-II (14) mice were maintained in specific pathogen-free conditions in the Department of Microbiology and Immunology, University of Melbourne, animal house following institutional guidelines. The gBT-I transgenic mice express a V
2/V
8.1+ TCR from a CTL clone (HSV-2.3) (15) that recognizes the HSV-1 gB498–505 determinant complexed with H-2Kb. The OT-I and OT-II mice express MHC class I- and II-restricted TCR (V2/V5.1+) specific for the OVA peptides OVA257–264 and OVA323–339, respectively. The peptides SSIEFARL (gB498–505), SIINFEKL (OVA257–264), and KISQAVHAAHAEINEAG (OVA323–339) were obtained from Auspep. The KOS strain of HSV-1 was propagated and titered using VERO cells grown in MEM plus 10% FCS. The rHSV-OVA strain expressing the OVA protein was made by homologous recombination with a packaging competent hybrid vector (ori-AAV/HSV) in the manner described (16).
T cell and DC cultures
Activated transgenic T cells from gBT-I, OT-I, or OT-II mice were generated in vitro by coculturing transgenic splenocytes with irradiated (3000 rad) C57BL/6 splenocytes, pulsed with the relevant peptide at 1 µg/ml (10–6 M). The cells were cultured for 4 days in the presence of IL-2 (500 U/ml) in RPMI 1640 plus 10% FCS, and LPS (0.25 µg/ml) was added to the OT-II cells for the final 2 days of culture. C57BL/6 spleen-derived DC were grown in vitro in our laboratory from spleen precursors in NIH 3T3 supernatant and GM-CSF, as described previously (17).
Adoptive transfers, virus infections, and DC immunizations
In vitro activated T cells (106 cells) were harvested and transferred into C57BL/6 mice. Twenty-four hours after transfer, mice were infected in each hind footpad with 4 x 105 PFU HSV-1 KOS or HSV-OVA in PBS. Before harvesting, DC cultures were treated with anti-CD40 Ab (FGK45) (18) at 50 µg/ml for 48 h, or LPS at 10 µg/ml for 24 h to mature the cells. On the day of transfer, DC were harvested (naive, LPS, or CD40 treated), and half the cells in each group were pulsed with gB498–505 peptide at 1 µg/ml for 45 min, 37°C. All cells (6 groups) were then labeled with CFSE (Molecular Probes) by incubating with CFSE (2.5 µM) at 37°C for 10 min. Viable cell counts were performed, and C57BL/6 mice were immunized in the footpad with 5 x 106 DC per foot.
mAbs and flow cytometry
The activation phenotype of in vitro activated gBT-I, OT-I, or OT-II T cells was determined using anti-CD8 allophycocyanin (53-6.7), anti-CD4 PE (GK1.5), anti-V2 FITC (B20.1), anti-CD25 PE (PC61), anti-CD44 PE (IM7), anti-CD62L PE (MEL-14), and anti-CD43 FITC (1B11) mAbs (BD Pharmingen), and Kb-gB498–505 or Kb-OVA257–264 tetramers. The maturation phenotype of cultured DC was determined before immunization by staining with anti-CD11c biotin (HL3) plus streptavidin allophycocyanin, anti-CD86 PE (GL1), and anti-I-Ab FITC (AF6–120.1) Abs obtained from BD Pharmingen. Forty-eight hours after immunization, single-cell suspensions were prepared from the PLN of DC-immunized mice and examined for CFSE+ cells using a BD Biosciences FACSort. Dead cells were excluded using propidium iodide. Statistical analysis was performed using Student’s t test (95% confidence).
Ex vivo CTL assay
Ex vivo CTL lysis was assessed by a 4-h chromium release assay with 51Cr (150 mCi)-labeled EL4 cells with or without gB498–505 peptide at 1 µg/ml. EL4 are a C57BL/6-derived (H-2b) MHC class II– thymoma cell line and were maintained in DMEM supplemented with 10% FCS. Single-cell suspensions prepared from the PLN of mice, immunized with DC 5 days earlier, were assayed at E:T ratios of 100:1. Statistical analysis was performed using Student’s t test (95% confidence).
In vivo CTL assay
In vivo CTL activity was determined, as described previously (19). Briefly, target cells were prepared from C57BL/6 splenocytes pulsed with 1 µg/ml gB498–505 peptide and labeled with a high concentration (2.5 µM) of CFSE (CFSEhigh population), or incubated without peptide and labeled with a low concentration (0.25 µM) of CFSE (CFSElow population). An equal number of cells from each population (107) was adoptively transferred into mice, which were sacrificed 4 h later. Cell suspensions were analyzed by FACS, and each population was distinguished by their different fluorescent intensities. Lysis was determined by loss of the peptide-pulsed CFSEhigh population from the PLN in relation to the peptide-negative CFSElow population.
APC assays
The ex vivo detection of APC was performed, as described previously, using the HSV-specific hybridoma HSV2.3.2E2 (19). Briefly, PLN were removed from mice infected with HSV or HSV-OVA in the footpad 2 or 5 days previously and treated with a collagenase/DNase solution (1 mg/ml collagenase type II (Worthington Biochemical) and 0.1% grade II bovine pancreatic DNase I (Boehringer Mannheim) in DMEM plus 10% FCS) for 20 min, followed by the addition of 50 µl of 0.1 M EDTA for a further 5 min. Two-fold serial dilutions of the cells were made in flat-bottom, 96-well plates starting at 106 cells/well. A total of 105 hybridoma cells was added to each well and cultured overnight. The 5-bromo-4-chloro-3-indolyl -D-galactoside (X-Gal) assays were performed on the cultures to identify the responding hybridomas by first washing the wells with PBS and fixing the cells with 100 µl of PBS containing 2% formaldehyde and 0.2% glutaraldehyde for 5 min at 4°C. After a further wash, 50 µl of a solution containing 1 mg/ml X-Gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 2 mM MgCl2 in PBS was added to each well. Cultures were examined microscopically for the presence of blue (LacZ+) cells after 8- to 12-h incubation at 37°C. Titrations of known numbers of gB-presenting APC were used as controls to ensure the number of LacZ+ cells accurately represented numbers of APC. Statistical analysis was performed using Student’s t test (95% confidence).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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A number of reports have demonstrated that CTL can lyse APC in an Ag-specific manner following injection of in vitro derived professional APC (7, 8, 9, 10). The adaptive immune response to cutaneous HSV infection has been shown to peak in the draining PLN 5 days after infection (20, 21, 22). Substantial numbers of armed CTL specific for the immunodominant epitope from HSV glycoprotein B (gB498–505) can be found in PLN at this time (23). We wanted to address whether HSV gB-presenting APC arising from this infection survive within the PLN in the face of these effector CTL. To ascertain the level of presentation, we used a CD8+ T cell hybridoma specific for gB498–505 that expresses -galactosidase under the control of the IL-2 promoter (24). LN cells from infected mice were cultured with the hybridoma cells overnight, which allows for sensitive detection of APC presenting the virus-derived Ag (19, 25). We have shown previously that this presentation is confined to the CD8+CD11C+ DC population within these tissues (26). In this manner, we determined that a significant number of APC-presenting gB498–505 are detectable in the PLN of normal C57BL/6 mice 5 days after cutaneous infection with HSV (Fig. 1A). Moreover, analysis of in vivo CTL activity within the PLN at this time demonstrated maximal killing of the CFSE-labeled targets (Fig. 1B) (23). Thus, during the normal course of the immune response, many APC-presenting specific MHC class I-peptide ligands are capable of surviving within an environment that contains abundant cytolytic activity.
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We next examined whether endogenous APC possess a constitutive ability to survive interactions with armed CTL effectors. We have shown previously that significant gB498–505 presentation is detectable in the PLN 48 h after infection with HSV (5, 19). However, few armed effectors are present at this early stage (23). As a consequence, we could examine what effect introduced CTL populations had on this endogenous presentation in the absence of significant endogenous T cell responses. Infection of control C57BL/6 mice showed greater numbers of gB-presenting APC were present in the PLN at this time (Fig. 2). To examine whether the presence of pre-existing CTL could eliminate such presentation, we adoptively transferred activated gBT-I CTL into mice before infection. These CTL were generated from CD8+ T cells specific for gB498–505 derived from H-2Kb-restricted TCR transgenic gBT-I mice (12). The gBT-I CTL, activated in vitro with peptide in the presence of IL-2, displayed a typical activated phenotype (CD25high, CD44high, CD43high, and CD62Llow), bound H-2Kb-gB tetramers, and demonstrated excellent CTL activity both in vitro and in vivo (data not shown). Following transfer, we observed a significant reduction in detectable Ag presentation in the PLN in comparison with control mice (Fig. 2). No detectable difference in viral clearance was observed in the tissues at this time (data not shown), suggesting that reduced Ag presentation in the PLN was not due to a reduction in virus in the tissues. Thus, the presence of pre-existing CTL dramatically reduced the number of endogenous gB-presenting APC in the draining LN following HSV infection, suggesting that these cells are not intrinsically resistant to CTL-mediated elimination.
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To enable the direct observation of APC elimination in vivo, we made use of DC lines, generated from splenic precursors cultured with GM-CSF (17). The DC were either pulsed with gB498–505 peptide or left untreated, and all were labeled with CFSE before injecting mice in the footpad with 1 x 106 cells per mouse. gBT-I CTL, or control OT-I CTL, were adoptively transferred into the mice, and 2 days later the draining PLN were removed and cell suspensions were analyzed by flow cytometry for the presence of CFSE+ DC. We could readily detect the CFSE-labeled DC in the draining LN of C57BL/6 mice, regardless of whether they had been pulsed with peptide before immunization (Fig. 3A). This is in agreement with the migration patterns of DC following immunization described by others (7, 8, 27). Most importantly, we found that gBT-I CTL abolished the appearance of resting DC within the PLN, while these same peptide-labeled DC survived in the presence of activated OT-I T cells (Fig. 3A).
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We next examined the immune response generated by immunization with peptide-pulsed DC, directly ex vivo using a standard 4-h 51Cr assay. At the numbers transferred in this study, the gBT-I CTL showed minimal direct ex vivo killing because of low numbers present within the PLN (1–2% of CD8+ T cells). Both resting and LPS-matured gB-presenting DC elicited comparable cytolytic activity in the PLN 5 days after immunization (Fig. 4). The response was noticeably stronger following immunization with CD40-matured DC, supporting the role for CD40 and T cell help in promoting immune responses (28, 29). Cotransfer of gBT-I CTL with resting peptide-labeled DC generated minimal CTL activity, correlating with elimination of the CFSE+ DC (Fig. 3). No such abrogation was observed in control mice that received OT-I CTL. Together these data demonstrate that APC survival and functional priming in vivo in the presence of specific CTL are enhanced upon maturation of the DC.
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The above results suggested that the gB-specific CD8+ T cells might exert their effects by killing gB-presenting APC, if such CTL were present at the early stages of the response. This raises the question of how such APC can survive in the presence of endogenous CTL generated during the normal progression of the response (see Fig. 1). Medema et al. (11) used an in vitro model to demonstrate that helper-derived signals can mature DC, inducing the expression of a granzyme B inhibitor that protects against CTL-induced lysis. This suggested that following in vivo infection, APC may initiate protective measures to survive interactions with CTL. To examine the role of Ag-specific CD4+ T cell help, we made use of a recombinant strain of HSV expressing the OVA protein (HSV-OVA). Effector CD4+ T cells were produced by in vitro peptide stimulation of OT-II transgenic T cells specific for the I-Ab-restricted OVA323–339 peptide. The effector OT-II CD4+ T cells proliferated extensively; up-regulated CD25, CD44, and CD43; and down-regulated CD62L (data not shown). Mice were adoptively transferred with combinations of effector gBT-I and OT-II T cells either alone or together, before infection with HSV-OVA or wild-type HSV via the footpad. Two days later, gB-presenting APC from the PLN were quantitated using the LacZ-inducible T cell hybridomas. An equivalent level of gB presentation was detected after HSV or HSV-OVA infection of mice transferred with OT-II cells alone or no cells (B6) (Fig. 5). The ability of gBT-I CTL to abolish gB498–505 presentation was established after both HSV and HSV-OVA infection. Following the cotransfer of gBT-I and OT-II cells, we were able to detect significant numbers of gB-presenting APC in the PLN after infection with HSV-OVA, but not HSV. This suggested that the CD4+ T cells were prolonging Ag presentation within the PLN in an Ag-specific manner. Together these results suggest that APC involved in the initiation of viral immunity can resist CTL-mediated attack as a consequence of signals derived from interactions with Th cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Detectable Ag presentation persists for at least 7 days following HSV infection (5), despite the presence of maximal CTL numbers in the PLN at this time (23). Given the relatively short t1/2 of MHC class I complexes on cell surfaces (30), this suggests that following cutaneous HSV infection, a steady influx of APC from the tissues may provide a continuous Ag source in the PLN. Nevertheless, removal of the site of infection, and thus this putative flow of Ag, 8 h after infection demonstrates that specific APC may survive for up to 4 days in vivo (5). Infections such as Sendai virus and influenza virus can involve presentation in the draining LN for 10 days to 4 wk, respectively, presumably in the presence of considerable numbers of CTL (6, 25). We show in this study that DC lysis resulted in the abrogation of CTL priming in the PLN, suggesting that, during HSV infection, APC are at least partially protected from such destruction.
We examined the role of CD4+ and CD8+ T cells in the survival of introduced APC in vivo and the effect this has on the resulting immune response. The benefits of sustained T cell-APC interactions in effector and memory generation suggest that APC ought to have a means of protecting themselves from CTL. It is known that DC are resistant to killing via Fas (CD95) (31), yet killing of APC can occur independent of either perforin or Fas (9). Evidence that maturation of DC results in up-regulation of the granzyme B inhibitor SPI-6 (11) suggests that APC are capable of surviving interactions with effector CD8+ T cells, at least in vitro. Evidence also suggests that DC are programmed to die, but T cell help may prolong their survival (32). We show in this study that Ag-specific CD4+ T cells can protect endogenous APC from CTL-mediated elimination during the course of a viral infection. This may constitute an important mechanism involved in the regulation of immune responses. Yet, the exact means by which Th cells protect are uncertain. Although it is likely that CD40L (CD154) is involved, other molecules such as Fas ligand, which, like CD40L, is up-regulated on the surface of effector CD4+ T cells (33, 34), have been shown to induce the maturation of DC (35). In support of this, we demonstrate that maturation signals alone, such as anti-CD40 stimulation or bacterial LPS, are sufficient to protect DC from elimination by CTL.
We show that CTL can remove both endogenous and introduced APC presenting the gB498–505 peptide, if they are present sufficiently early during the immune response. This supports previous reports showing that killing of DC can occur in vivo (7, 8, 9, 10). The loss of peptide-pulsed DC correlated with a lack of CTL priming within the PLN, suggesting that the gBT-I CTL were killing APC either in the PLN or within the tissues themselves. A recent report by Ronchese and colleagues (36) suggested that DC killing occurs within the tissues, not the LN, because DC were not killed if present in the LN before the transfer of CTL. We cannot rule out that gBT-I CTL simply held the gB-presenting APC in the tissues or prevented their entry into the PLN. We feel this is unlikely, however, given that LPS- and CD40-matured DC were able to migrate into the PLN, despite the presence of the gBT-I CTL.
In the course of a normal immune response, both CD4+ and CD8+ T cells are activated in concert, thus allowing helper-APC interactions to occur before expression of lytic capability by CD8+ T cells. Protecting the life span of an APC, however briefly, may have important consequences to immunity if a greater number of naive cells can be activated. Clearly, some level of APC killing would be expected to occur, particularly as the number of maturing CTL reaches a critical threshold. This may serve to limit the duration of Ag presentation, as has been suggested for Listeria monocytogenes infection (37), although differences in the pathogen or the magnitude of the helper response may define the extent of presentation. Nonetheless, it is likely that there exists a balance between APC survival and death based on the degree of interaction between CD4+ and CD8+ T cells. In addition, the phenomenon of APC killing may have more of a role in secondary responses, whereby rapid responses by pre-existing CTL can inhibit other similar or cross-reactive specificities (38, 39, 40).
In summary, we demonstrate that endogenous APC-presenting viral peptides following HSV infection can be killed in an Ag-specific manner by pre-existing CTL in vivo. Addition of CD4+ T cells can prolong the survival of gB-presenting APC following HSV infection. In addition, maturation of DC via LPS or anti-CD40 protected against CTL lysis in vivo, promoting the priming of an anti-gB response, whereas CTL-induced loss of the resting DC suppressed this immunity. This may have important implications for our understanding of the control of immune responses, in particular the significance of CD4+ T cell-derived help in both promoting and optimizing immune responses to viral infection.
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Disclosures |
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Footnotes |
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1 This work was supported in part by grants from the National Health and Medical Research Council of Australia. 
2 Current address: Emory Vaccine Center and Department of Microbiology and Immunology, Emory University, Atlanta, GA 30322.
3 Address correspondence and reprint requests to Dr. Francis R. Carbone, Department of Microbiology and Immunology, University of Melbourne, Parkville, Australia VIC 3010. E-mail address: fcarbone{at}unimelb.edu.au
4 Abbreviations used in this paper: LN, lymph node; DC, dendritic cell; gB, glycoprotein B; PLN, popliteal LN; X-Gal, 5-bromo-4-chloro-3-indolyl -D-galactoside.
Received for publication January 19, 2006. Accepted for publication March 28, 2006.
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References |
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- and -chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76: 34-40. [Medline]
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M. D. H. Lay, L. Zhang, R. M. Ribeiro, S. N. Mueller, G. T. Belz, and M. P. Davenport Kinetics of Major Histocompatibility Class I Antigen Presentation in Acute Infection J. Immunol., January 15, 2009; 182(2): 902 - 911. [Abstract] [Full Text] [PDF]
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M. Y. Gerner, K. A. Casey, and M. F. Mescher Defective MHC Class II Presentation by Dendritic Cells Limits CD4 T Cell Help for Antitumor CD8 T Cell Responses J. Immunol., July 1, 2008; 181(1): 155 - 164. [Abstract] [Full Text] [PDF]
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C. Baron, M.-C. Meunier, E. Caron, C. Cote, M. J. Cameron, D. J. Kelvin, R. LeBlanc, V. Rineau, and C. Perreault Asynchronous Differentiation of CD8 T Cells That Recognize Dominant and Cryptic Antigens J. Immunol., December 15, 2006; 177(12): 8466 - 8475. [Abstract] [Full Text] [PDF]
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