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The Cross-Priming APC Requires a Rel-Dependent Signal to Induce CTL¹

Justine D. Mintern^*,, Gabrielle Belz^*, Steve Gerondakis^*, Francis R. Carbone^2, and William R. Heath^2,*,

^* Immunology Division, Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria, Australia; Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia; and Cooperative Research Center for Vaccine Technology, Queensland Institute of Medical Research, Royal Brisbane Hospital, Herston, Australia

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Induction of OVA-specific CTL by cross-priming requires help from CD4 T cells, which use CD154 to signal CD40 on the APC. To further dissect the molecular pathways involved in cross-priming, we examined the role of Rel, an NF-B family member. c-rel^-/- mice failed to generate OVA-specific CTL by cross-priming, but could induce CTL to HSV-1. Using chimeric mice, Rel expression was shown to be required by the APC, but not by the T cells. Notably, the deficiency in Rel could be overcome by triggering CD40, implying that the APC required Rel before receipt of the CD40 signal. These data suggest that the cross-priming APC must receive two signals before it can stimulate CTL. The first signal is Rel dependent and is required before activation of CD4 helper T cells, which then deliver the second signal using CD154 to trigger CD40.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cross-priming involves the capture and processing of exogenous cell-associated Ags into the class I Ag presentation pathway (1). This form of priming has been demonstrated for the generation of CTL responses to graft tissues (2), tumor cells (3), and intracellular pathogens such as viruses (4). To dissect the cellular and molecular signals involved in cross-priming, our studies have focused on the induction of OVA-specific CTL after injection of irradiated spleen cells loaded with whole OVA protein (5, 6). We have shown that CTL generation is dependent on 1) host bone marrow-derived APC, which are responsible for capturing OVA from the irradiated spleen cells and presenting it to the T cells (5), and 2) CD4 Th cells, which up-regulate CD154 and trigger CD40 on the APC, converting it to a cell fully competent for CTL priming (6). Recently, the CD8⁺ dendritic cell was identified as the APC responsible for the generation of OVA-specific CTL by cross-priming (7).

To identify the molecular pathways involved in cross-priming, we have begun to examine CTL induction in mice defective in specific signal transduction pathways. Since studies have shown that triggering CD40 can lead to NF-B activation (8, 9), we tested CTL generation in mice deficient for Rel, one of the members of the NF-B family of transcription factors (reviewed in Refs. 10, 11). NF-B transcription factors normally reside as inactive forms in the cytoplasm until upon appropriate stimulus, Rel/NF-B dimers are translocated to the nucleus. Signals shown to elicit Rel/NF-B translocation include those downstream of TNFR family members, Toll-like receptor (TLR)³ family members, and Ag receptors on both T and B cells. These pathways result in the expression of numerous immunologically relevant genes, including those encoding chemokines, cytokines, and other inflammatory mediators. Thus, Rel/NF-B activation plays a central role in innate and acquired immunity (reviewed in Ref. 12).

Rel, the first identified member of the NF-B transcription factor family, can function as a homodimer or form heterodimers, with RelA, NF-B1, or NF-B2 (10). Its expression is mostly restricted to hemopoietic cells such as B cells, T cells, neutrophils, and macrophages (reviewed in Ref. 13). Dendritic cells, including the splenic CD8⁺ dendritic cell (25), also express Rel. Rel-deficient (c-rel^-/-) mice show relatively normal hemopoiesis; however, in vitro proliferation of T and B cells is poor in response to mitogens (8, 14). Despite this, infection of c-rel^-/- mice with influenza virus leads to normal CTL immunity with only slightly delayed viral clearance (15). In this report, we examined the role of Rel in the generation of CTL by cross-priming.

	Materials and Methods

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Mice

All mice were used between 6 and 12 wk old and were bred and maintained at the Walter and Eliza Hall Institute for Medical Research. OT-II (16), Rag-1-deficient OT-I mice (17), and c-rel^-/- mice (8) have been previously described. c-rel^-/- mice were backcrossed to C57BL/6 for 10 generations. Rag-1-deficient OT-I mice were backcrossed to C57BL/6 for 8 generations

OVA-specific CTL generation

For priming mice with cell-associated OVA, syngeneic bm1 spleen cells were washed in HEPES-buffered Eagle’s medium plus 2.5% FCS (HF2.5), irradiated at 1000 cGy, centrifuged, and then incubated at 2 x 10⁸ cells/ml with 10 mg/ml OVA in HEPES-buffered Eagle’s medium for 10 min at 37°C. After three washes in HF2.5, 20 x 10⁶ cells were injected i.v. into mice. In contrast to our previous reports, OVA was not loaded by osmotic shock, but simply added to cells for the 10-min incubation period. This has been shown to cross-prime CTL immunity (18). Seven days after priming, OVA-specific CTL were restimulated in vitro as previously described (5). Briefly, spleens were removed and single cells were cultured with 1500 cGy irradiated OVA_257–264-coated B6 spleen cells (10⁸) for 6 days in 30 ml RPMI 1640 (mouse tonicity) with 10% FCS, 2-ME, glutamine, and antibiotics. Cytotoxicity was assessed in a conventional ⁵¹Cr release assay using the H-2^b cell line EL4 with and without OVA-peptide coating as targets. Percent OVA-specific lysis = (lysis of peptide-coated EL4 targets) - (lysis of EL4 targets).

HSV-specific CTL generation

To generate HSV-specific CTL, mice were primed with 10⁵ PFU of HSV-1 i.v. After 7 days, spleens were removed and single cells were cultured with 1500 cGy irradiated HSV glycoprotein B-peptide (gB_498–505)-coated B6 spleen cells (10⁸) for 6 days in 30 ml RPMI 1640 (mouse tonicity) with 10% FCS, 2-ME, glutamine, and antibiotics. Cytotoxicity was assessed in a conventional ⁵¹Cr release assay using the H-2^b cell line EL4 with and without gB_498–505 coating as targets. Percent HSV-specific lysis = (lysis of peptide-coated EL4 targets) - (lysis of EL4 targets).

Generation of bone marrow chimeras

To generate bone marrow chimeras, adult mice were lethally irradiated with 900 cGy and reconstituted with a total of 5 x 10⁶ T cell-depleted bone marrow cells of the appropriate background. The next day, mice were injected with 100 µl of T24 (anti-Thy-1) ascites to deplete radioresistant T cells. These mice were then left at least 8 wk before immunization. Reconstitution was confirmed by using flow cytometry to analyze the peripheral blood.

Reconstitution of Rag-1-deficient mice

Rag-1-deficient mice were injected i.v. with 20 x 10⁶ c-rel^-/- or B6 lymph node cells. Mice were then left 2 wk before immunization.

Preparation of responder T cells

OT-I and OT-II cells were prepared as described elsewhere (19, 20). Briefly, OT-I cells were derived from the spleen and lymph nodes of Rag-1-deficient OT-I mice. These cells were treated with RL172 (anti-CD4) and J11d (anti-heat-stable Ag) for 30 min on ice, centrifuged, and then depleted by treatment with rabbit complement for 30 min at 37°C. For OT-II cells, lymph nodes were removed from OT-II mice and single cells were treated with 3.168 (anti-CD8) and J11d and then complement as above. The percentage of V2⁺CD8⁺ cells (for OT-I) or V2⁺CD4⁺ cells (for OT-II) was determined by flow cytometry using a FACScan (BD Biosciences, Mountain View, CA) as previously described (19, 20).

Fluorescent labeling of T cells

CFSE labeling was performed as previously described (21). Briefly, semipurified T cells (OT-I or OT-II) were resuspended in PBS containing 0.1% BSA (Sigma-Aldrich, St. Louis, MO) at 10⁷ cells/ml. For CFSE labeling, 1 µl of CFSE (Molecular Probes, Eugene, OR) stock solution (5 mM in DMSO) was incubated with 10⁷ cells for 10 min at 37°C. Cells were washed twice in HF2.5, counted, and resuspended at the appropriate concentration in HF2.5 for injection.

Priming mice in the presence of an anti-CD40 stimulus

OVA-specific CTL generation was performed as described above. Stimulation via CD40 was performed by injecting the mice for 4 consecutive days with 100 µg of (anti-CD40) FGK45 (IgG2a) i.v., beginning on the day of immunization with OVA-coated spleen.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
c-rel^-/- mice do not generate OVA-specific CTL by cross-priming

To determine whether Rel has a role in the induction of CTL by cross-priming, normal B6 mice and c-rel^-/- mice were primed with 20 x 10⁶ OVA-coated irradiated spleen cells (OVA-coated spleen) i.v., and 7 days later their spleen cells were restimulated in vitro and assessed for OVA-specific CTL activity (Fig. 1a). This revealed that c-rel^-/- mice could not generate OVA-specific CTL by cross-priming. CTL immunity was not universally impaired in c-rel^-/- mice, however, as they have been reported to respond to influenza virus (15), and in our hands were able to respond to HSV (Fig. 1b).

View larger version (13K): [in this window] [in a new window]   FIGURE 1. c-rel-/- mice do not generate OVA-specific CTL by cross-priming. B6 mice (•) or c-rel-/- mice () were immunized i.v. with 20 x 106 irradiated OVA-coated bm1 spleen cells (a) or 105 PFU of HSV-1 (b). Seven days after priming, the spleen cells from each mouse were restimulated in vitro for 6 days and then examined for their ability to lyse 51Cr-labeled EL4 targets alone or pulsed with the appropriate peptide. Percent OVA- or HSV-specific lysis represents peptide-dependent lysis. Nonspecific EL4 lysis was below 6%. The data presented are representative of nine experiments with one to two mice per group for OVA-coated spleen priming, and two experiments with two mice per group for HSV priming.

Failure of c-rel^-/- mice to generate CTL could have been due to either an APC defect or a T cell defect (or a combination of both). To determine whether CD4 and CD8 T cells also needed to express Rel for effective cross-priming, Rag-1-deficient mice were adoptively transferred i.v. with c-rel^-/- lymph node cells to produce mice with normal APC (from the Rag-1-deficient host) and c-rel^-/- CD4 and CD8 T cells. When these mice were primed with OVA-coated spleen they generated OVA-specific CTL immunity (Fig. 2), showing that T cells do not require Rel for cross-priming.

View larger version (18K): [in this window] [in a new window]   FIGURE 2. T cells do not require Rel for cross-priming. Rag-1-deficient mice were injected i.v. with 20 x 106 B6 (•) or c-rel-/- () lymph node cells. Two weeks later, the mice were primed with 20 x 106 irradiated OVA-coated bm1 spleen cells. Seven days after priming, the spleen cells from each mouse were restimulated in vitro for 6 days and then examined for their ability to lyse 51Cr-labeled OVA254–267-pulsed or unpulsed EL4 targets. Nonspecific EL4 lysis was below 15%. Percent OVA-specific lysis represents peptide-dependent lysis. The data presented are representative of two experiments with two to three mice per group.

The above data suggest that the T cells were functional in the absence of Rel and therefore the inability to cross-prime was due to an APC defect. To formally address whether c-rel^-/- APC were capable of cross-priming, CTL induction was examined in chimeric mice that contained normal T cells and c-rel^-/- APC. These were generated by reconstituting B6 mice with a combination of two different types of bone marrow: one was c-rel^-/- H-2^b bone marrow, which possessed the correct MHC haplotype to present OVA to CD8 T cells, i.e., K^b, but lacked Rel; the second was c-rel^+/+ H-2^bm1 bone marrow, which will make normal T cells but possessed the wrong MHC haplotype (K^bm1) for its APC to present OVA to CD8 T cells (22). When these mixed bone marrow chimeras were primed with OVA-coated spleen, they failed to generate CTL immunity (Fig. 3a). This was not due to a failure in reconstitution, because they responded effectively to HSV (Fig. 3b). These data suggested that c-rel^-/- mice could not be cross-primed because they required Rel expression in their APC.

View larger version (13K): [in this window] [in a new window]   FIGURE 3. c-rel-/- mice do not generate OVA-specific CTL due to an APC-specific defect. (B6 x bm1)F1 mice were lethally irradiated and reconstituted with B6 (•) or an equal ratio of bm1 and c-rel-/- () bone marrow. Eight weeks later, the mice were immunized i.v. with 20 x 106 OVA-coated bm1 (a) or 105 PFU of HSV-1 (b) . Seven days later, their spleens were removed and stimulated in vitro for 6 days before being tested in a 51Cr release assay using EL4 targets alone or pulsed with the appropriate peptide. Percent OVA- or HSV-specific lysis represents peptide-dependent lysis. Nonspecific EL4 lysis was below 15%. The data presented are representative of five experiments with one to two mice per group for OVA-coated spleen priming and two experiments with two mice per group for HSV priming.

The above data indicated that Rel expression was required in the APC, but it was unclear whether it participated in APC development or a function associated with the priming phase of the response. To determine whether Rel was essential for the development of the cross-priming APC, we examined Ag presentation in vivo. c-rel^-/- mice were injected with either OT-I or OT-II cells that had been labeled with CFSE. These mice were then primed with OVA-coated spleen. Three days after priming, spleens of recipient mice were removed and single cells were analyzed by flow cytometry (Fig. 4). The strong proliferative response of both OT-I and OT-II T cells indicated that the cross-priming APC was able to develop in c-rel^-/- mice and could present OVA to both CD4 and CD8 T cells. It is important to note that although the proliferation of OT-I and OT-II cells suggests that the c-rel^-/- APC are capable of "priming" both T cell subsets, other studies (23) indicate that in vivo proliferation of transgenic T cells as shown in Fig. 4 does not always correlate with the ability to prime a naive normal repertoire. In fact, while low frequencies of OVA-specific T cells, such as those found in a normal repertoire, require CD4 T cell help for priming, the high frequencies used in the above adoptive transfer experiments will respond in a helper-independent manner. Thus, the helper requirements for CTL induction are bypassed in these studies. So, although these data in Fig. 4 are not informative about the capacity of the c-rel^-/- APC to prime a normal repertoire, they clearly indicate that in the absence of Rel, the cross-priming APC is able to develop from bone marrow precursors and then capture and present Ag.

View larger version (27K): [in this window] [in a new window]   FIGURE 4. c-rel-/- APC develop normally and can present Ag. B6 or c-rel-/- mice were injected with CFSE-labeled OT-I or OT-II transgenic T cells. Mice were left untreated or the next day primed with 20 x 106 irradiated OVA-coated bm1 spleen cells. Three days later, the mice were sacrificed and spleen cells were analyzed by flow cytometry. Profiles are gated on CD8+, CFSE+, propidium iodide-negative cells and include all cells of this phenotype obtained from one-tenth of a host spleen. The data presented are representative of three experiments with two to three mice per group.

We have previously shown that cross-priming requires CD4 T cell help, which is mediated by triggering of CD40 on the APC, licensing it for priming naive CTL. Given that Rel is reported to be essential for CD40-stimulated B cell proliferation (8, 9), we investigated whether Rel also functioned downstream of CD40 signaling within the cross-priming APC. This was achieved by priming c-rel^-/- mice in the presence of a CD40-stimulatory mAb, FGK45 (Fig. 5). We expected that if Rel was involved in the CD40 signal, FGK45 would be inactive in c-rel^-/- mice. Surprisingly, c-rel^-/- mice generated strong CTL immunity in the presence of a CD40 stimulus. Therefore, Rel was not essential downstream of CD40 engagement and must act before this point in the response. Furthermore, these data make two additional important points: 1) confirmation that the cross-priming APC develops normally in c-rel^-/- mice and 2) that OVA-specific CD8 T cells do not require Rel to respond.

View larger version (16K): [in this window] [in a new window]   FIGURE 5. CD40-specific mAb treatment replaces the requirement for Rel in the generation of OVA-specific CTL. c-rel-/- mice were immunized i.v. with irradiated OVA-coated bm1 spleen cells and left untreated (•) or injected i.v. daily for 4 days with either 0.1 mg of a CD40-specific mAb FGK45 (). Seven days after priming, the spleen cells from each mouse were restimulated in vitro for 6 days and then examined for their ability to lyse 51Cr-labeled OVA257–264-pulsed or unpulsed EL4 targets. Percent OVA-specific lysis represents peptide-dependent lysis. The data presented are representative of four experiments with one to three mice per group. Priming c-rel-/- mice with OVA-coated spleen in the presence of an isotype control mAb did not induce OVA-specific CTL.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Currently, the only identified signaling pathway required for cross-priming is that mediated via CD40 (6). We expected Rel to be involved in this signal. Interestingly, the inability of c-rel^-/- APC to cross-prime was rescued when the APC were activated via CD40 (Fig. 5). Therefore, in the cross-priming APC, in contrast to B cells, CD40 signaling does not require Rel, at least for generating signals for CTL priming. This supports the observation that events downstream from CD40 differ in these cell types (26). More importantly, the ability of a CD40 signal to overcome the requirement for Rel in the cross-priming APC implies that the Rel and CD40-mediated signals are independent. Therefore, these data support a model where the cross-priming APC requires two signals to elicit successful CTL priming.

The origin of the Rel-dependent signal is unknown, but most likely comes from the Ag. A variety of upstream molecules are known to signal nuclear translocation of Rel. One likely candidate pathway is that mediated by TLRs, which are known to activate Rel/NF-B when engaged (reviewed in Ref. 27). Since signaling through TLRs has been reported to be triggered by a wide variety of pathogen products (28, 29, 30) as well as heat shock proteins (31), there is ample potential for such signals to be supplied by our priming material. Alternatively, Rel is reported to act in the transmission of signals downstream of TNF- receptors I and II (10). Therefore, TNF- induced as a consequence of priming-associated inflammation may also be responsible for the Rel-dependent signal.

A potential role for Rel in the cross-priming APC is signaling the up-regulation of CD40 expression. This is supported by the observation that pathogen-mediated signals increase the expression of CD40 on dendritic cells (32). Therefore, in the absence of Rel, CD40 expression may be insufficient to enable APC activation by CD4 Th cells. This possibility is unlikely, however, since anti-CD40 Ab is able to bypass the requirement for Rel, suggesting that a functional level of CD40 expression can be achieved by Rel-deficient APC.

An alternative model is one where Rel acts at a step before CD40-mediated activation of the cross-priming APC. Given that the CD40 signal is delivered by CD40 ligand (CD154) expressed by activated CD4 Th cells (6, 33, 34), we suggest that in the absence of Rel, CD4 T cell activation is inadequate. We propose that the Rel-dependent signal is required for the cross-priming APC to sufficiently stimulate CD4 T cell up-regulation of CD154. In support of this model is the observation that c-rel^-/- mice can generate CTL immunity to HSV (Fig. 1) and influenza virus (15), where neither CTL response requires CD4 T cell help (35, 36). Licensing of the APC with viral products that overcome the requirement for CD4 T cell activation renders the Rel-dependent signaling pathway unnecessary. Therefore, CTL responses to influenza and HSV are Rel independent. Signaling via Rel may also directly aid APC priming of the naive CD8 T cells; however, clearly this is not essential.

Several potential Rel-transcribed genes may contribute to the activation of CD4 T cells including cell adhesion molecules and the MHC class II molecule itself (reviewed in Ref. 13). The production of cytokines such as IL-2 (37) and IL-12 (25) are also shown to be Rel dependent. The cross-priming CD8⁺ dendritic cell is reported to be the major dendritic cell subset responsible for IL-12 production (38). It is plausible that in the absence of Rel, the failure of the cross-priming APC to generate IL-12 may hamper the activation of CD4 T cells and consequently limit their ability to signal through CD40. It is currently under investigation as to whether IL-12 is required for cross-priming. Finally, Rel is implicated in the expression of chemokine receptors and chemokines that allow trafficking of APC into T cell areas or attract T cells to Ag-carrying APC (39). Therefore, in the absence of Rel, CD4 T cells may not interact optimally with the APC, limiting delivery of the essential CD40 signal.

In summary, we propose a model where the APC must receive two signals before it can induce CTL immunity by cross-priming. One signal must result in the nuclear translocation of Rel. We propose that the Ag itself provides the Rel-dependent signal but that this signal alone is insufficient to elicit cross-priming. Once triggered by the Rel-dependent signal, the APC is capable of activating CD4 Th cells causing the up-regulation of CD154. Consequently, the cross-priming APC receives a second signal via CD154 and is now sufficiently licensed to enable successful priming of naive CTL and the generation of CTL immunity.

	Acknowledgments

 
We thank Tatiana Banjanin, Freda Karamalis, Loretta Clovedale, Jane Langley, and Annette Alafacci for their technical assistance.

	Footnotes

 
¹ This work was funded by National Institutes of Health Grant AI43347-01 and grants from the National Health and Medical Research Council of Australia and a Howard Hughes Medical Institute International Fellowship.

² Address correspondence and reprint requests to Dr. William R. Heath, Immunology Division, Walter and Eliza Hall Institute, P.O. Royal Melbourne Hospital, Parkville 3050, Victoria, Australia. E-mail address: heath{at}wehi.edu.au, or Dr. Francis R. Carbone, Department of Microbiology and Immunology, University of Melbourne, Parkville 3050, Victoria, Australia. E-mail address: f.carbone{at}microbiology.unimelb.edu.au

³ Abbreviation used in this paper: TLR, Toll-like receptor.

Received for publication October 25, 2001. Accepted for publication January 28, 2002.

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