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Key Words: virus • infection • cytotoxic T lymphocyte • dendritic cell
Abbreviations used: DCs, dendritic cells; L, ligand; LCMV, lymphocytic choriomeningitis virus; MFI, median fluorescence intensity; VSV, vesicular stomatitis virus.
Induction of CTL responses after immunization with minor histocompatibility antigens, such as the HY antigen, requires the presence of Th (1, 2). CTL responses induced by cross-priming, i.e., by priming with exogenous antigens that reached the class I pathway, have also been shown to require the presence of Th (3). These observations have important consequences for tumor therapy, where Th may be critical for the induction of protective CTL responses by tumor cells (4). An important effector molecule on activated Th is the CD40 ligand (L)1 interacting with CD40 on B cells, macrophages, and dendritic cells (DCs) (5). Triggering of CD40 on B cells is essential for isotype switching and the generation of B cell memory (5). More recently, it was shown that stimulation of CD40 on macrophages and DCs leads to their activation and maturation (6, 7). Specifically, DCs upregulate costimulatory molecules and produce cytokines, such as IL-12, upon activation. Interestingly, this CD40L-mediated maturation of DCs seems to be responsible for the helper effect on CTL responses. In fact, it has recently been shown that CD40 triggering by Th renders DCs able to initiate a CTL response (8–10). This is consistent with the earlier observation that Th have to recognize their ligands on the same APC as the CTLs, indicating that a cognate interaction is required (3). Thus, CD40L-mediated stimulation by Th leads to the activation of DCs, which subsequently are able to prime CTL responses.
In contrast to these Th-dependent CTL responses, viruses are often able to induce protective CTL responses in the absence of Th (for review, see reference 11). Specifically, lymphocytic choriomeningitis virus (LCMV) (12–16), vesicular stomatitis virus (VSV; reference 17), influenza virus (18), vaccinia virus (19), and ectromelia virus (20) were able to prime CTL responses in mice depleted of CD4+ T cells or deficient for the expression of class II or CD40. The mechanism for this Th-independent CTL priming by viruses is not presently understood. Moreover, most viruses do not completely stimulate Th-independent CTL responses, but virus-specific CTL activity is reduced in Th-deficient mice. Thus, Th may enhance antiviral CTL responses, but the mechanism of this help is not yet fully understood. DCs have recently been shown to present influenza-derived antigens by cross-priming (21). It is therefore possible that, similar to the mechanism shown for minor histocompatibility antigens and tumor antigens (8–10), Th may assist induction of CTLs via CD40 triggering on DCs.
This study analyzes the mechanism of Th-independent versus Th-dependent CTL priming triggered by viruses. We found that CD40 is not measurably involved in the induction of CTLs specific for LCMV or VSV. Thus, although VSV-specific CTL responses are partly dependent upon the presence of CD4+ T cells (17), this helper effect was not mediated by CD40L. However, the virus-specific CTLs were observed to solicit their own help in vivo by themselves triggering a maturation process in DCs. This maturation of DCs occurred in complete absence of viral infection upon injection of specific peptide in a TCR-transgenic mouse model. The degree of Th independence of the CTL responses induced by the two different viruses was paralleled by a correspondingly efficient maturation of DCs. Interestingly, CD8+ T cells not only mediated activation of the DCs they were interacting with but also triggered DC maturation in trans, leading to widespread activation of DCs in lymphoid organs. Thus, in contrast to minor histocompatibility antigens that only induce abortive CTL responses in the absence of Th, viruses nucleate a sufficiently strong CTL response that leads to generalized activation of DCs, rendering the response independent of Th.
The LCMV isolate WE was provided by Dr. P. Ohashi, Toronto, Canada and grown on L cells at a low multiplicity of infection. VSV serotype Indiana was originally provided by R.M. Zinkernagel (Institute of Experimental Immunology, Zürich, Switzerland) and grown on BHK cells at a low multiplicity of infection.
Detection of LCMV- and VSV-specific CTL In Vitro.
In Vivo Expansion and Effector Cell Induction.
To assess cytolytic effector function, spleen cells were tested in 51Cr-release assays using peptide p33 (KAVYNFATM)-pulsed EL-4 target cells. To distinguish endogenous CTL activity of the C57BL/6 recipient mice from CTL activity of the transferred TCR-transgenic T cells, EL-4 cells pulsed with peptide MB6 (KAVVNIATM) were also used as target cells. MB6 is recognized by the TCR-transgenic T cells but not by the polyclonal C57BL/6 T cells (29).
Isolation and Characterization of DC.
In Vivo–induced DC Maturation by Peptide.
For the T cell stimulation assay, DCs were sorted using a FACStarPLUSTM, obtaining a purity >97%. Different numbers of sorted DCs (H-2b) were added to 2 x 104 purified T cells obtained from spleens of H-2d BALB/c (MLR) or female HY-transgenic mice (H-2b on a RAG-2 background) and cultured in 96-well plates (Falcon; Becton Dickinson) for 3 d. T cell proliferation was assessed by [3H]thymidine (1 µCi/well) uptake in a 16-h pulse after 72 h.
In Vitro Maturation of DC.
Generation of Chimeric Mice.
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Mice and Viruses.
Mice deficient for the expression of CD40 (22), MHC class II (23), β2-microglobulin (24), and RAG-2 (25) have been described previously. Mice exhibiting a mutation in their H-2Db molecule (B6.C–H-2bm13) were provided by Dr. P. Ohashi, Ontario Cancer Institute, Toronto, Canada. Transgenic mice expressing a TCR specific for peptide p33 derived from LCMV presented in association with H-2Db and transgenic mice expressing a TCR receptor specific for the HY antigen have been described previously (26, 27). Mice were bred under specific pathogen-free conditions according to Swiss federal law. Class II–/– mice were back-crossed on a C57BL/6 background, and C57BL/6 mice were used as controls. CD40+/– mice were used as controls for CD40–/– mice.
Mice were immunized intravenously with LCMV (200 PFU) or VSV (2 x 106 PFU), and 8 or 6 d later, respectively, spleen cell suspensions were prepared and tested directly in a 51Cr-release assay, using LCMV-derived peptide p33 (KAVYNFATM)- or VSV-derived peptide (SDLRGYVYQGLKSG)-pulsed EL-4 cells as target cells (28). Lytic units were calculated for a 50% level of cell lysis. As different control mice were used for CD40–/– versus class II–/– mice, results are expressed as percent lytic units of controls.
Spleen cells from TCR-transgenic control mice (106 cells) were adoptively transferred into normal C57BL/6 recipient mice. 2 h later, mice were challenged with LCMV. After 8 d, spleen cells were harvested and stained for CD8 (PE; PharMingen) and transgenic V
2 expression (FITC; PharMingen), and the presence of TCR-transgenic cells was assessed by flow cytometric analysis.
Mice were immunized intravenously with LCMV (200 PFU) or VSV (2 x 106 PFU). 6 or 5 d later, respectively, spleens of uninfected and virus-infected mice were dissected, cut into pieces, and digested twice with collagenase D (Boehringer Mannheim) for 30 min at 37°C in a shaking water bath. Cells were recovered by centrifugation and resuspended in an OptiprepTM (Nycomed Pharma) gradient as previously described (30). Low density cells were then incubated for 30 min on ice with FITC-labeled anti-CD11c (1:400; PharMingen) and with PE-labeled anti–B7-1, anti–B7-2, anti-CD40, and isotype control, in the presence of 5% normal mouse serum. Cells were washed and analyzed by a FACSCaliburTM flow cytometer (Becton Dickinson), excluding propidium iodide– positive cells. Percent upregulation of median fluorescence intensity (MFI) was calculated as follows: % upregulation = ([MFI induced – MFI control]/MFI control – 1) x 100.
Transgenic mice expressing a TCR specific for LCMV were intravenously injected with 10 µg LCMV-derived peptide p33 (KAVYNFATM), A4Y (KAVANFATM), V4Y (KAVVNFATM), or S4Y (KAVSNFATM). After 24 h, DCs were isolated from spleens of treated and, as control, untreated mice and monitored for expression of B7-1, B7-2, and CD40 as described above. Activation of T cells was assessed by measuring CD69 (PE) expression on CD8+ T cells (FITC).
CD8+ T cells were positively selected from mesenteric LNs of a RAG-2–/– LCMV-specific, TCR-transgenic mouse by anti-CD8–coated magnetic beads (GmbH; Miltenyi Biotec). DCs (105 cells/well) obtained from control B6, CD40–/–, and MHC class II–/– mice were pulsed for 1 h with 10 µg/ml peptide p33, A4Y, V4Y, or S4Y, washed, and cocultured with 3 x 105 naive CD8+ T cells at 37°C. After 20 h, DCs were double-stained with FITC-labeled anti-CD11c and PE-labeled anti–B7-1, anti–B7-2, and anti-CD40 and analyzed by a FACSCaliburTM flow cytometer, excluding propidium iodide–positive cells.
RAG-2–/– mice were irradiated (3 Gy) and reconstituted with 5 x 106 bone marrow cells from CD45.1 congenic C57BL/6 mice and 5 x 106 bone marrow cells derived from CD45.2 TCR-transgenic B6.C–H-2bm13 mice. 10 wk later, recipient mice exhibited large numbers of TCR-transgenic T cells and a mixed DC population of both CD45.1 and CD45.2 allotypes. DCs were isolated 24 h after injection of peptide or saline as described above, triple-stained with APC-labeled anti-CD11c, FITC-labeled anti-CD45.2, and PE-labeled B7-2, anti-CD40, or negative control, and analyzed by using a FACSCaliburTM flow cytometer.
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CD40 Is Not Involved in Virus-specific Th-dependent and -independent CTL Responses.
To compare the role of CD4+ T cells versus the presence of CD40 for the induction of LCMV-specific CTL responses, control, CD40–/–, and MHC class II–/– mice that lacked Th were immunized with LCMV. At the peak of the response, i.e., 8 d after infection, spleen cells were harvested and tested in a 51Cr- release assay. To be able to quantify the relative numbers of CTL effector cells in the different mice, lytic units were calculated (Fig. 1). Absence of MHC class II or CD40 did not affect the frequency of CTL precursor after infection with LCMV.
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CD40 and MHC class II have been implicated in thymic selection (23, 31). We therefore wanted to exclude the possibility that alterations in the CTL populations in CD40–/– or MHC class II–/– mice were responsible for the Th independence of the CTL response. Thus, T cells from transgenic mice expressing a TCR specific for LCMV (26) were adoptively transferred into the different mice before viral infection. It has been shown previously that this leads to a dramatic expansion of transgenic T cells (32). 8 d after infection, at the peak of the antiviral response, presence of T cells expressing the transgene-encoded TCR (V2) was assessed in the spleens of the mice. The absolute number and the frequency of transgenic T cells was comparable in control, CD40–/–, and MHC class II–/– mice (Fig. 2, A and B). Moreover, a comparable lytic activity of the transgenic cells was observed in a 51Cr-release assay on peptide p33–pulsed target cells (Fig. 2 C). Similar results were obtained with peptide MB6, which is selectively recognized by the transgene-encoded TCR (data not shown). These results demonstrate that a defined population of CTLs expressing a single TCR can be stimulated by LCMV in the absence of both CD40 molecules and CD4+ Th.
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chains, T cells derived from HY–TCR-transgenic mice on a RAG-2–/– background were used. As expected for a Th-dependent CTL response, control DCs stimulated only low-level proliferation of specific T cells. In contrast, CTL-activated DCs derived from p33-primed animals triggered much stronger proliferation of specific T cells (Fig. 6 B). Thus, CTL-mediated activation of DCs rendered Th-dependent CD8+ T cell responses largely Th independent.
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Viruses Trigger Th-independent CTL Responses.
It has been known for some time that viruses are able to stimulate protective CTL responses in the absence of CD4+ T cells (12– 20). However, CTL responses are impaired in the absence of Th after infection with poorly replicating viruses such as VSV (17). To date, it has not been analyzed whether CD4+ T cells assist CTLs via a CD40L-dependent mechanism under these circumstances. This study demonstrates that this is not the case. Thus, even the Th-dependent part of a virus-specific CTL response is not dependent upon the CD40–CD40L interaction. Compared with tumor-specific CTL responses, where Th assist CTL induction by activating DCs via CD40 triggering, the Th-dependent production of cytokines, such as IL-2, seems to be more important during viral infections. This interpretation is consistent with the previous observation that cytokine secretion by Th after viral infection is not impaired in the absence of CD40 (39).
DC Maturation Triggered by CD8+ T Cells.
A large proportion of DCs isolated from virally infected mice exhibited a mature phenotype. This maturation of DCs occurred in the absence of Th and CD40. Various stimuli are known to activate DCs. Specifically, microbial components, such as LPS, bacterial DNA, or cell walls; inflammatory cytokines; and CD40 triggering are able to stimulate expression of costimulatory molecules on DCs and induce their migration to central lymphoid organs (6, 7). Moreover, it has been shown that LPS is able to activate splenic DCs in vivo and trigger the migration of DCs from the red pulp to T cell areas (40). Interestingly, infection of splenic APCs by influenza virus directly induces the expression of costimulatory molecules and enhances the immunogenicity of DCs (8, 41). Thus, it was possible that the observed Th-independent maturation of DCs was (a) directly mediated by viral components, (b) mediated by non-T cells such as NK cells, or (c) mediated by CD8+ T cells. To address this question, various gene-deficient mice were infected with LCMV, and the maturation of DCs was assessed. Maturation of DCs occurred in class II–/– as well as class I–/– mice. These results demonstrated that DC maturation could be mediated by either CD4+ or CD8+ T cells. DCs isolated from transgenic mice expressing a class I–restricted TCR 24 h after injection of specific peptide also exhibited an activated phenotype. This demonstrated that DC maturation triggered by CD8+ T cells could occur in complete absence of viral infection. Interestingly, the maturation stimulus delivered by CD8+ T cells was not dependent upon a direct interaction of CD8+ T cells with DCs. In contrast, CD8+ T cells could activate DCs in trans, and DCs that failed to present the relevant peptide due to a mutation in the class I molecule also underwent maturation in TCR-transgenic mice injected with peptide. Thus, freshly activated CD8+ T cells are able to induce generalized activation of DCs, even in the absence of direct T cell–DC contact.
There are parallels between the activation of DCs by CD8+ T cells described here and the IFN system. Type I IFNs induce an antiviral state in many different cell types. This antiviral state is induced in trans, i.e., virally infected cells are able to stimulate neighboring cells via secretion of type I IFNs. Similarly, CD8+ T cells induce an immunostimulatory state, not only in virally infected DCs that present the relevant peptides but also in neighboring APCs. However, the two systems are apparently not identical, because (a) type I IFNs induce an antiviral rather than an immunostimulatory state in DCs and (b) DC activation by CD8+ T cells occurs in the absence of functional type IFN receptors (data not shown).
Using the in vitro system of CD8+ T cell–mediated DC activation and T cells or DCs derived from various gene-deficient mice together with neutralizing antibodies and other blocking molecules, we tried to define the factor responsible for the observed maturation of DCs. So far, we can exclude maturation mediated by CD28, CD40L, TRANCE, TNF, IL-1, IL-4, IL-6, IL-17, IFN-/β, and IFN-
, but have not yet identified the critical molecule. Future studies will therefore address the cloning of the as yet elusive factor.
Th-independent CTL Responses: a Threshold Phenomenon?
It has been observed that viruses are able to generate CTL responses in the absence of Th (12–20). Interestingly, those viruses that induced strong CTL responses in the presence of Th also did so in the absence of Th, suggesting that the Th dependence of the response may simply be determined by the overall strength of the response. Along a similar line, recombinant viral proteins injected in association with insect cell debris as an adjuvant were able to induce very strong CTL responses by cross-priming (42), and this cross-priming occurred in the absence of Th (42). Thus, the failure of model antigens to induce CTL responses in the absence of Th is not absolute but rather seems to be determined by the strength of the response. The results presented here may provide an explanation for these findings by suggesting that generation of CTL responses may occur as a threshold phenomenon due to a positive feedback mechanism. Accordingly, virus-infected DCs are able to activate a few naive CTLs. These activated T cells in turn secrete inflammatory cytokines/chemokines that may lead to the activation of neighboring APCs. The number of CD8+ T cells that can be activated in such a way determines the efficiency of DC activation. Thus, if only few CD8+ T cells are activated initially, the response may be abortive, as too few DCs undergo maturation. In contrast, if a sufficient number of CTLs is triggered, widespread activation of DCs may occur, and an immunostimulatory program is initiated in lymphoid organs that renders the response independent of Th. Thus, during tumor-specific responses or upon cross-priming, few CD8+ T cells are activated, and the response remains abortive in the absence of Th, whereas during antiviral immune responses, a sufficient number of CD8+ T cells becomes triggered to render the response Th independent. In this model, VSV may represent an intermediate case. It induces only weak upregulation of costimulatory molecules in vivo. Correspondingly, CTL responses partly depend on the presence of Th.
Interestingly, the requirements for activation of DCs were very stringent. Peptide A4Y, which is a relatively weak agonist that nevertheless stimulates efficient proliferation of specific T cells in vitro, almost completely failed to trigger the activation program in DCs in vivo. Thus, only T cells interacting with strong agonists are able to stimulate maturation of DCs, offering an explanation for why many model antigens that are often weak agonists compared with viral peptides require Th for induction of CTL responses.
This study demonstrates that CD8+ T cells can mediate activation of DCs in complete absence of viral infection. However, it should be noted that a cross-talk between the innate and specific immune systems during viral infection may also facilitate the induction of Th-independent CTL responses. Thus, although CD8+ T cells can solicit their own help by themselves inducing maturation of DCs, other factors are likely to contribute to the efficiency of antiviral CTL responses (11).
Autoimmunity Triggered by Viruses: Are Activated DCs the Missing Link?
Viral infections are thought to be an important cause for the induction or exacerbation of autoimmune diseases (43). It has been argued that cross-reactive T cells are responsible for disease, and it has been shown that viral peptides can stimulate autoreactive T cell clones (44, 45). Moreover, there is good evidence that Herpes Simplex virus causes autoimmune stromal keratitis by activating self-specific, cross-reactive T cells (46). However, cross-reactive T cells are not always the critical factor for disease. In the case of Coxsackie virus–induced diabetes, it has been shown that the disease is mediated by T cells recognizing self-antigens that do not cross-react with viral proteins. These self-antigens were apparently released from lysed virus-infected cells and subsequently activated the self-specific T cells (47). However, presence of these antigens alone may not be sufficient for the induction of self-specific T cells, because low amounts of most self-antigens also reach lymphoid organs in the absence of infection due to physiological cell turnover (48). Thus, the presentation of self-antigens in lymphoid organs per se does not seem to be responsible for the induction of autoimmunity, likely because immature DCs in lymphoid organs that process those antigens most efficiently are inefficient at stimulating naive T cells. However, the nonspecific maturation of DCs and the concomitant upregulation of costimulatory molecules triggered by the antiviral immune response may be responsible for the activation of self-specific T cells that usually ignore their antigens in vivo. The generalized activation of DCs upon viral infection may therefore not only boost virus-specific T cell responses and render them independent of Th but may also shift the balance from ignorance to autoimmunity.
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Submitted: 11 December 1998
Revised: 3 March 1999
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| TABLE OF CONTENTS |
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, control; •, CD40–/–; 
, class II–/–.
) or control animals (