By
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From the * Basel Institute for Immunology, CH 4005 Basel, Switzerland; the Institute for
Experimental Immunology, 8019 Zürich, Switzerland; and the § Laboratory of Cellular Physiology and
Immunology, the
Laboratory of Immunology, and the ¶ Howard Hughes Medical Institute,
The Rockefeller University, New York 10021
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Abstract |
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CD40 ligand (CD40L), a tumor necrosis factor (TNF) family member, plays a critical role in
antigen-specific T cell responses in vivo. CD40L expressed on activated CD4+ T cells stimulates antigen-presenting cells such as dendritic cells, resulting in the upregulation of costimulatory molecules and the production of various inflammatory cytokines required for CD4+ T cell
priming in vivo. However, CD40L- or CD40-deficient mice challenged with viruses mount
protective CD4+ T cell responses that produce normal levels of interferon , suggesting a
CD40L/CD40-independent mechanism of CD4+ T cell priming that to date has not been elucidated. Here we show that CD4+ T cell responses to viral infection were greatly diminished
in CD40-deficient mice by administration of a soluble form of TNF-related activation-induced
cytokine receptor (TRANCE-R) to inhibit the function of another TNF family member,
TRANCE. Thus, the TRANCE/TRANCE-R interaction provides costimulation required
for efficient CD4+ T cell priming during viral infection in the absence of CD40L/CD40. These results also indicate that not even the potent inflammatory microenvironment induced
by viral infections is sufficient to elicit efficient CD4+ T cell priming without proper costimulation provided by the TNF family (CD40L or TRANCE). Moreover, the data suggest that
TRANCE/TRANCE-R may be a novel and important target for immune intervention.
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Introduction |
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ATNF family member, CD40L, has been shown to be
critical for the generation of antigen-specific T cell
responses in vivo (1). CD40L expressed on activated
T cells triggers CD40 on macrophages and dendritic cells
(DCs),1 resulting in the upregulation of costimulatory molecules and the induction of IL-12 in these APCs (9).
These costimulatory molecules and IL-12 then potentiate
CD4+ T cell responses in vivo (1, 9, 10, 14). However, CD40L- or CD40-deficient mice challenged with
viral infections such as lymphocytic choriomeningitis virus
(LCMV) are able to mount protective CD4+ T cell responses that produce normal levels of IFN- (19). In
addition, a majority of CD40L-deficient patients with hyper-IgM syndrome (HIGM) do not show increased susceptibility to various infections associated with defective CD4+ T
cell immune responses (22), suggesting that some pathogens are able to activate CD4+ T cells via a CD40L/CD40-independent pathway. The mechanism determining CD40/
CD40L independence of CD4+ T cell responses during intracellular infections is not understood. It has been speculated
that destruction of infected cells and the production of various inflammatory cytokines (e.g., INF-
) in response to, for
example, viral infections constitute a sufficiently powerful adjuvant effect to allow the activation of T cells in the absence
of costimulation provided by CD40L/CD40 interaction.
In this study, the cellular immune responses to viral infections were examined in order to elucidate the factor(s) responsible for CD40L/CD40-independent CD4+ T cell priming. We show here that CD40L/CD40-independent activation of CD4+ T cells during viral infection requires TRANCE/TRANCE-R interaction (23). Thus, this study suggests that CD4+ T cell priming in general is likely to be regulated by one of two TNF family members (CD40L or TRANCE).
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Materials and Methods |
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Mice, Viruses, Cells, Abs, and Recombinant Proteins.
CD40-deficient mice have been described and were originally provided by Dr. H. Kikutani (Osaka University, Osaka, Japan [26]). As control mice, CD40+/Infection and Treatment with TR-Fc.
For LCMV-specific CD4+ T cell proliferation, mice were infected intravenously or into one hind footpad with 200 PFU of LCMV WE. Spleen cells were isolated 13 or 30 d later, and proliferation and cytokine production were measured as described (19). To assess cytotoxicity, mice were infected intravenously with 200 PFU of LCMV, and spleen cells were isolated 8 d later. For influenza virus-specific proliferation, mice were infected intranasally with virus (0.1 hemagglutination U/mouse). Spleen cells were isolated 8 d later. Mice were injected three times, on days 0, 2, and 5 after infection, with 100 µg of either TR-Fc or control hIgG1.CTL and B Cell Responses.
EL-4 target cells were pulsed with peptide p33 (KAVYNFATM) at a concentration of 10In Vitro Proliferation and Production of IFN-.
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Results and Discussion |
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A recently identified member of the TNF receptor family, TRANCE-R (also called RANK), has been shown to be expressed at high levels on mature DCs (23). Moreover, TRANCE treatment enhanced the survival of mature DCs, indicating that TRANCE-R may exhibit a similar function as CD40 on these cells (24). To test whether TRANCE/ TRANCE-R interaction may play a role in T cell activation, surface expression of TRANCE was analyzed on activated T cells. Similar to CD40L, surface TRANCE expression was highly upregulated on T cells upon stimulation through antigen receptors (Fig. 1 A). Moreover, when mature DCs were treated with soluble TRANCE, the expression of IL-12 (Fig. 1 B) and other inflammatory cytokines (e.g., IL-1 or IL-6; data not shown) was induced in mature DCs, a property also shared by CD40L (11). Together, these results suggested that TRANCE and CD40L may share some similar functions in vivo during T cell activation and that TRANCE may be responsible for CD40L-independent CD4+ T cell responses, as observed in some murine model systems such as during viral infections (19).
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To test this hypothesis, we chose to study the immune response to LCMV infection as a murine model since it has been extensively characterized and also because the activation of CD4+ T cells during LCMV infection was shown not to be affected in CD40L- or CD40-deficient mice (19).
To analyze whether TRANCE is upregulated in vivo during the course of an immune response after viral infection, mice were infected with LCMV, and spleen cells were analyzed for TRANCE expression 8 d later. Indeed, the proportion of TRANCE-expressing T cells increased after infection (~6% of CD4+ T cells and ~7% of CD8+ T cells became TRANCE-positive, whereas 0% of T cells expressed TRANCE in uninfected control mice).
To determine whether TRANCE plays a role during immune responses in vivo, and if so, whether it exhibits a compensatory role for CD40L during viral infections, we tested
the consequences of blocking the TRANCE/TRANCE-R
interaction by injection of TR-Fc on antigen-specific B,
CD8+, and CD4+ T cell responses induced by LCMV infection in control (C57BL/6 or CD40+/) and CD40-deficient mice (26).
The most prominent role of CD40L is to promote isotype switching in activated B cells and to allow the formation of germinal centers (GCs; 1-3). Indeed, CD40-deficient mice failed to produce high titers of LCMV-specific IgG Abs and produced no GCs (Fig. 2, A, D, and E). In contrast, TR-Fc-treated C57BL/6 mice mounted LCMV-specific IgG responses comparable to those of control mice treated with hIgG1 (Fig. 2 A) and generated similar numbers of GCs of normal architecture (Fig. 2, B and C). These results suggest that the TRANCE/TRANCE-R interaction does not play a critical role in T-B cell collaboration, despite the low level of TRANCE-R that can be detected on activated B cells (data not shown).
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We next analyzed the ability of TR-Fc-treated control and CD40-deficient mice to mount LCMV-specific T cell responses. Mice were injected with LCMV, and CD8+ T cell-mediated responses were analyzed in a 51Cr-release assay 8 d later (Fig. 3 A). In keeping with previous reports (20, 21), the CD40L/CD40 interaction was not required for efficient primary CTL responses against LCMV (Fig. 3 A). In addition, inhibition of the TRANCE/TRANCE-R interaction did not affect the LCMV-specific acute CTL responses (Fig. 3 A). Moreover, inhibition of both the TRANCE/ TRANCE-R and CD40L/CD40 interactions did not affect acute CTL responses (Fig. 3 A). These results suggest that primary LCMV-specific CTL responses are largely independent of CD40L and TRANCE on activated T cells.
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LCMV-specific CD4+ T cell responses were then examined early after infection (day 13) in TR-Fc-treated control
and CD40-deficient mice by measuring in vitro recall proliferative responses. As reported previously (19), LCMV-specific CD4+ T cells produced a Th1 cytokine pattern, since large
amounts of IFN- (Fig. 3 C) but not IL-4 (data not shown)
were detected in culture supernatants. Purified CD4+ T cells
from CD40-deficient mice proliferated normally and produced, although at reduced levels, IFN-
after stimulation
with LCMV-derived antigens (Fig. 3, B and C), indicating
that LCMV can prime antigen-specific CD4+ T cells in a
CD40L/CD40-independent manner. TR-Fc-treated control mice also mounted normal CD4+ T cell responses (Fig.
3, B and C). In marked contrast, the proliferative response
of CD4+ T cells in TR-Fc-treated CD40-deficient mice
was nearly completely blocked (Fig. 3 B). In addition, the
production of IFN-
was also completely abrogated in
these mice (Fig. 3 C). This was not due to immune deviation, since blocking the TRANCE/TRANCE-R interaction in control or CD40-deficient mice did not upregulate
IL-4 production (data not shown). To determine whether
the lack of LCMV-specific CD4+ T cell responses was due
to a delay in T cell priming in the absence of both CD40L/
CD40 and TRANCE/TRANCE-R interactions, CD4+
T cells were purified and restimulated with viral antigens
1 mo after infection. Even 30 d after infection, no significant LCMV-specific CD4+ T cell responses were detected
in the absence of both CD40L/CD40 and TRANCE/
TRANCE-R interactions (Fig. 4). Therefore, the results indicate that either the CD40L/CD40 or the TRANCE/
TRANCE-R interaction is required for induction of CD4+
T cell responses by LCMV.
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To analyze whether TRANCE can also mediate CD40L/ CD40-independent CD4+ T cell responses in other viral systems, CD40-deficient mice were infected with influenza virus, and virus-specific CD4+ T cell responses were analyzed (Fig. 5). As observed for LCMV, influenza virus can prime antigen-specific CD4+ T cells in a CD40L/CD40-independent manner, and the induction of virus-specific CD4+ T cell responses was greatly inhibited in the TR-Fc-treated CD40-deficient mice (Fig. 5). Thus, TRANCE/TRANCE-R provides a major costimulatory stimulus in the absence of CD40L/CD40 for CD4+ T cell responses to influenza viruses.
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In summary, this study establishes the TRANCE/ TRANCE-R interaction as an important player in CD4+ T cell responses in vivo. Moreover, we also show that the TRANCE/TRANCE-R interaction compensates for a lack of CD40L/CD40 interaction to allow efficient CD4+ T cell responses during viral infection. This explains why viruses can induce CD4+ T cell immune responses in CD40L- or CD40-deficient mice. In addition, this study also shows that despite the destruction of infected cells and the production of various inflammatory cytokines in response to viral infection, efficient CD4+ T cell priming still requires costimulation predominantly by TNF family members (i.e., either TRANCE or CD40L), which is analogous to CD4+ T cell priming induced by purified proteins administered with CFA (in this case, costimulation provided by CD40L). Therefore, it is possible that CD4+ T cell priming in general may require costimulation by at least one TNF family member (e.g., CD40L or TRANCE).
CD40L-mediated CD4+ T cell activation occurs indirectly via activation of the APCs (1, 9). Specifically,
in vitro stimulation of CD40 on DCs stimulates a maturation process culminating in the upregulation of costimulatory molecules and the capacity to produce IL-12, a cytokine important for production of IFN- by CD4+ T cells
(1, 9). Although stimulation of TRANCE-R on mature DCs fails to upregulate costimulatory molecules on these
cells (24), we showed that, similar to CD40L, TRANCE
treatment triggered generation of IL-12 and other proinflammatory cytokines by mature DCs. In addition, when
stimulated in vitro by anti-CD3, purified T cells proliferated and produced normal levels of cytokines in the presence of TR-Fc (data not shown), suggesting that, similar to
the CD40L/CD40 interaction, there is no direct role for the TRANCE/TRANCE-R interaction in T cells. Therefore,
the TRANCE/TRANCE-R and CD40L/CD40 interactions between CD4+ T cells and APCs may have functional
consequences primarily for the APCs, e.g., promoting DC
viability and cytokine production (1, 9, 24). It is presently not known why some antigens (e.g., proteins in adjuvants) use predominantly the CD40L-dependent pathway (1) while others (e.g., viruses [this study]) use both TRANCE- and CD40L-dependent pathways of CD4+
T cell stimulation. It is possible that certain viruses directly upregulate TRANCE-R during DC differentiation. Alternatively, there may be different requirements for induction
of TRANCE and CD40L on T cells. It is also possible, although not yet determined, that some pathogens might use
predominantly the TRANCE-dependent pathway to elicit
efficient CD4+ T cell responses. The CD40L/CD40 interaction is an important site for manipulating the immune response in order to facilitate organ transplantation and to reduce atherosclerosis (1, 29), and our in vivo findings now
suggest that the interaction of TRANCE and its receptor
may be an additional target for immunotherapy.
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Footnotes |
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Address correspondence to Martin F. Bachmann, Basel Institute for Immunology, Grenzacherstrasse 487, CH 4005 Basel, Switzerland. Phone: 41-61-605-1228; Fax: 41-61-605-1364; E-mail: bachmann{at}bii.ch; or to Yongwon Choi, Laboratory of Immunology, The Rockefeller University, 1230 York Ave., New York, NY 10021. Phone: 212-327-7441; Fax: 212-327-7319; E-mail: choi{at}rockvax.rockefeller.edu
Received for publication 8 January 1999.
The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche, Basel, Switzerland. This work was supported in part by National Institutes of Health Medical Scientist Training Program grant GM-07739 (to B.R. Wong), and National Institutes of Health grants AI-44264 (to Y. Choi), AI-13013 (to R.M. Steinman), and AI-39672 (to R.M. Steinman). R. Josien is supported by a fellowship from the Revson Foundation. Y. Choi is an investigator of the Howard Hughes Medical Institute.We thank Manfred Kopf and Marco Colonna for comments and critical discussion. We also thank Angela Santana, Hong-Li Li, and Barbara Ecabert for excellent technical assistance.
Abbreviations used in this paper DC, dendritic cell; GC, germinal center; LCMV, lymphocytic choriomeningitis virus; TRANCE, TNF-related activation-induced cytokine.
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