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* Department of Microbiology and Immunology, Emory Vaccine Center, Emory University, Atlanta, GA 30329;
Department of Animal Sciences, University of Missouri, Columbia, MO 65211; and
Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isolated DC are prone to spontaneous apoptosis, and they can be rescued by factors such as GM-CSF, TNF-
, or TGF-
1 (4, 5, 6, 7), which induce antiapoptotic pathways mediated by bcl-2 and bcl-xL (8, 9). Furthermore, the addition of TNF-related activation-induced cytokine (TRANCE) and CD40L to cultures improves the survival of DC, correlating with an increased expression of bcl-xL and bcl-2, respectively (10, 11, 12). Analysis of the gene expression profile in a DC cell line showed that activation with Escherichia coli induced a rapid up-regulation of bcl-x levels, peaking at 12–18 h after stimulation and returning to baseline at 48 h (13). In contrast, mRNA levels of another antiapoptotic protein, bcl-2, decreased as early as 4 h after activation. Collectively, these data suggest that the maintenance of mature DC survival is likely to involve bcl-x genes; however, it has yet to be tested in vivo.
The bcl-x gene encodes multiple isoforms, including antiapoptotic bcl-xL and proapoptotic bcl-xS (14). bcl-xL localizes to the mitochondrial membrane, where it antagonizes the ability of bax or bak to release cytochrome c from the mitochondrion and induce a caspase-mediated cascade leading to cell death (15). Unlike the antiapoptotic bcl-xL, the proapoptotic bcl-x isoform bcl-xS lacks the C-terminal 189 bp of exon 2 due to the use of an alternative splice site (16). bcl-x knockout mice die at embryonic day 13, with signs of apoptosis in immature neurons of the brain, and in hemopoietic cells in the liver (17), prompting the generation of cre-lox conditional bcl-x knockout mice (bcl-xfl/fl) (18, 19).
To evaluate the role of bcl-xL in DC function, we used biolistic delivery of cre recombinase-encoding DNA vaccines to selectively knock out the bcl-x gene in skin-resident DC of bcl-xfl/fl mice. In conjunction, we also used RNA interference technology to specifically silence the bcl-xL isoform in DC. Our studies show that the antiapoptotic isoform bcl-xL is critical for the survival of Ag-bearing DC in secondary lymphoid tissues. bcl-xL-deficient DC underwent extensive apoptosis, and failed to induce protective immune responses in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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bcl-xfl/fl mice were imported from the University of Missouri, and analyzed for genotype by PCR as previously described (18, 19). C57BL/6 mice were obtained from Charles River Labs (Wilmington, MA). Mice used for experiments were between 4 and 10 wk of age, and maintained under standard pathogen-free housing conditions at the Emory Vaccine Center vivarium with the approval of the Institutional Animal Care and Use Committee at Emory University.
PCR and RT-PCR analyses
PCR identification of the recombined bcl-x locus in bcl-xfl/fl mice was performed as previously described (18, 19). To detect bcl-xL and bcl-xS isoforms, primers flanking the C-terminal 189 bp of exon 2 were used in a standard RT-PCR: forward primer, 5'-GGAGAGCGTTCAGTGATC-3', and reverse primer, 5'-CCAGCCACAGTCATGCC-3'.
DNA vaccine plasmids
Cre recombinase cDNA coupled with a SV40-derived nuclear localization sequence was cloned downstream of the CMV immediate-early promoter and upstream of a bovine growth hormone polyadenylation sequence (pCMV-Cre). Similarly, plasmids expressing full-length OVA cDNA (pCMV-OVA) and influenza hemagglutinin (HA) cDNA (pCMV-HA) were constructed. To track Ag-bearing cells, the lacZ gene was cloned downstream of the chicken -actin (CAG) promoter (pCAG-lacZ). To generate dual-expression DNA vaccines, these Ag expression cassettes were excised and blunt-end ligated into the cre-expressing vector. Dual-expression bcl-xL small interfering RNA (siRNA) plasmids were assembled by cloning the U6-bcl-xL-siRNA constructs downstream of pCAG-lacZ. Plasmids encoding pBcl-xS, pBcl-xL, and the mt7 pBcl-xL mutant were kind gifts from T. W. Behrens (University of Minnesota, Minneapolis, MN), S. Korsmeyer (Harvard University, Boston, MA), and J. M. Hardwick (Johns Hopkins University, Baltimore, MD), respectively.
Immunizations
Gene gun immunizations were performed on shaved abdominal skin of mice as described previously (3, 20). Briefly, a hand-held Helios gene delivery system (Bio-Rad, Hercules, CA) was used to deliver two doses of 0.5 µg of plasmid DNA coated on 1 mg of gold beads (DeGussa-Huls, Ridgefield Park, NJ) at a helium pressure of 400 psi. In experiments designed to study DC at the immunization site, we immunized ears of mice with one shot each on the dorsal and ventral sides (0.5 µg of plasmid DNA per shot) using the Helios gene gun set at a helium pressure of 600 psi.
T cell proliferation
Mice were immunized with pCMV-OVA, pCMV-Cre-CMV-OVA, or empty DNA vector, and draining superficial inguinal lymph nodes were removed and pooled 5 days later. CD11c+ cells were enriched to >90% purity by anti-CD11c (N418)-coupled magnetic beads (Miltenyi Biotec, Auburn, CA). Responder T cells were isolated from spleens of OT-II TCR transgenic mice by incubation of the single-cell suspension with anti-CD3-FITC (eBiosciences, San Diego, CA) Ab, followed by positive selection with anti-FITC microbeads (Miltenyi Biotec). CD11c+ DC from immunized mice were irradiated with 5000 rad and plated at a 1:1 ratio with OT-II T cells with irradiated accessory feeder cells. In control wells, OT-I (OVA257–264) and OT-II (OVA323–339) peptides were added to wells with OT-II T cells. Cells were pulsed with 1 µCi of [3H]thymidine after 48 h of culture, and assayed for radionucleotide incorporation in a MicroBeta plate reader (Wallac, Turku, Finland) the following day.
Serum ELISA
At days 15 and 30 after gene gun immunization, serum samples were collected from 
50 µl of blood obtained from mice via retro-orbital bleeds, and assayed on ELISA plates coated with sucrose gradient-purified A/PR/8/34 (H1N1) influenza virus. Quantitation of Ab levels was performed by comparison with a standard curve using goat anti-mouse IgG (BD Biosciences, San Jose, CA), and data were analyzed with Microplate Reader software (Bio-Rad).
Influenza challenge
Forty days following DNA vaccination with constructs expressing influenza HA, animals were challenged with a mouse-adapted influenza virus A/PR/8/34 (H1N1), a kind gift from H. Robinson (Emory University, Atlanta, GA). Allantoic fluid containing influenza virus was serially diluted in sterile PBS with 0.2% BSA (Sigma-Aldrich, St. Louis, MO), and a 50-µl volume containing 3x LD50 (0.03 HAU) viral dose was given intranasally to anesthetized mice. Following viral challenge, total body weight and survival were recorded over the subsequent 10 days.
siRNA constructs and testing
Candidate siRNA oligos targeted against the 189-bp region at the 3' end of exon 2 of murine bcl-x were devised according to guidelines established by Tuschl (21). The siRNA oligos were synthesized and cloned into the pSilencer 1.0 vector under control of the U6 RNA Polymerase III promoter per manufacturer’s instructions (Ambion, Austin, TX). Of the two siRNA constructs tested, the greatest knockdown was seen with the oligo targeting bcl-x468–488 (5'-AAGGAGATGCAGGTATTGGTG-3'). Ability to silence bcl-xL was tested by transient cotransfection with expression vectors encoding murine bcl-xL or bcl-xS into human 293 cells. The control GFP siRNA oligo was constructed following Sui et al. (22).
Epidermal cell preparation
To isolate epidermal cell suspensions, we excised the immunized ears at the base, and split-thickness ears were treated with 0.5% trypsin for 30 min at 37°C. Following incubation, a single-cell suspension was made by disrupting epidermal sheets in a 70-µm cell strainer.
Flow cytometry
For flow cytometric analysis of -galactosidase (-gal) expression, collagenase-treated lymph node cells or epidermal cell suspensions from ear skin were hypotonically loaded with 0.5 mM fluorescein di-D-galactopyranoside (Molecular Probes, Eugene, OR), as previously described (23, 24, 25). Aliquots of fluorescein di-D-galactopyranoside-loaded cells were stained with fluorochrome-conjugated Abs (anti-CD11c-allophycocyanin, -CD86-biotin, -I-Ab-PE, -annexin V-PE; BD Biosciences), with a secondary streptavidin-PerCP (BD Biosciences) Ab staining when needed. Cells were then acquired without fixation with a FACSCalibur (BD Biosciences), and data were analyzed with FlowJo software (Tree Star, Ashland, OR). We sorted cells to >94% purity with a FACSVantage (BD Biosciences), isolating CD11c+ cells from superficial inguinal lymph nodes of a naive C57BL/6 mouse, as well as CD11c+-gal+ and CD11c+-gal– populations from MACS-enriched CD11c+ cells from dLN of bcl-xfl/fl mice immunized 2.5 days earlier with pCMV-Cre-CAG-lacZ.
Statistical analyses
Statistical differences between experimental groups were analyzed with Prism software (GraphPad, San Diego, CA), with values of p < 0.05 from Student’s t test considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously, we had permanently marked and tracked the migration of Ag-bearing DC from the periphery to dLN by biolistic delivery of cre-encoding plasmids into a cre-indicator mouse strain, ROSA26R (3). We showed that DC resident in the skin could be directly transduced in vivo via gene gun immunization. In this study, we used the same approach to permanently delete the bcl-x gene from the genome of cells in bcl-xfl/fl mice. These mice have been engineered with two loxP sites flanking the bcl-x gene; in the presence of bacteriophage P1-derived cre recombinase, homologous recombination occurs between these two loxP sites, resulting in the permanent deletion of all bcl-x exons from the genome. bcl-xfl/fl mice received, via gene gun delivery to the abdomen, a DNA vaccine encoding cre recombinase (pCMV-Cre). The immunization site was marked, and then excised 6 h later and subjected to genomic DNA extraction. As controls, the same mouse received immunizations at nonoverlapping sites with either pCMV-OVA or gold particles alone. We then analyzed the genomic DNA samples for cre-mediated deletion of the bcl-x gene by PCR, using a primer set that produces a 150-bp fragment only from a deleted bcl-x locus (18, 19). Amplification of the 150-bp recombination product was restricted to the area of skin that had been immunized with cre recombinase (Fig. 1a). The adjacent areas of skin that were immunized with pCMV-OVA or empty gold particles did not produce the 150-bp recombination product, indicating that we could achieve a targeted, cre-dependent deletion of the bcl-x gene. The presence of two copies of the undeleted bcl-x gene was verified by PCR in all control DNA samples (data not shown).
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-gal. In these vectors, the strong hybrid CMV enhancer and CAG promoter drove expression of -gal to permit tracking of the small percentage of transfected cells. At 2.5 days after immunization, both the CD11c+-gal+ and the CD11c+-gal– populations were sorted from draining superficial inguinal nodes, and subjected to genomic DNA extraction and PCR amplification for the 150-bp recombination product. The CD11c+-gal+, but not the CD11c+-gal– population exhibited the 150-bp recombination product (Fig. 1b), indicating that biolistic delivery of the dual-expression plasmid deleted the bcl-x gene in skin-residing DC, which could then be tracked to the dLN. Decreased T cell stimulation by bcl-x-deficient DC
We first examined the effect of bcl-x deletion on the ability of skin-derived DC to stimulate naive T cell proliferation in vitro. We constructed a DNA vaccine that dually expresses cre recombinase and full-length chicken OVA (pCMV-Cre-CMV-OVA). We immunized cohorts of bcl-xfl/fl mice with this DNA vaccine, or with pCMV-Cre, pCMV-OVA, or an empty vector. Five days later, we magnetically enriched for CD11c+ DC from dLN, and used them directly in vitro as APCs, without addition of exogenous peptide, to stimulate proliferation of naive OVA-specific TCR transgenic CD4+ T cells (Fig. 2). CD11c+ DC isolated from mice immunized with pCMV-Cre-CMV-OVA had significantly reduced Ag-specific T cell proliferation, yielding a lower level of [3H]thymidine uptake than that achieved from immunization with pCMV-OVA alone (2.05 ± 0.1 vs 18.32 ± 0.6 x 103 cpm, respectively). Responder T cells pulsed with cognate OT-II peptide showed maximal amounts of proliferation (25.45 ± 2 x 103 cpm), whereas cells pulsed with control OT-I peptide, or stimulated with cells from animals immunized with either an empty vector or pCMV-Cre, showed only background levels of [3H]thymidine uptake. These data suggest that loss of the bcl-x gene in Ag-bearing DC resulted in a drastic loss of their T cell-priming capacity.
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To determine whether humoral immune responses would be blunted by the loss of bcl-x in Ag-bearing DC, we used the mouse influenza infection model system. Protection from influenza infection depends upon the generation of Abs against the viral HA protein (26). DNA vaccines encoding HA induce long-lasting and high titers of specific Abs (27). We immunized groups of bcl-xfl/fl mice with a DNA vaccine coexpressing cre recombinase and influenza HA (pCMV-Cre-CMV-HA), or with controls pCMV-HA or empty vector. At days 15 and 30 postimmunization, we collected sera from each mouse and measured the concentration of anti-HA Abs by a standard ELISA (Fig. 3a). Mice immunized with pCMV-HA exhibited 24.37 ± 2.4 µg/ml anti-HA IgG at day 15, and 77.39 ± 7.4 µg/ml at day 30 after vaccination. In contrast, mice immunized with pCMV-HA-CMV-Cre produced significantly lower levels of influenza-specific Abs at both time points (7.23 ± 2.6 and 9.3 ± 3 µg/ml; p < 0.05), whereas the response of control mice immunized with an empty DNA vector remained at baseline levels.
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bcl-x is needed for survival of DC
The loss of immunostimulatory capacity in DC established by the loss of bcl-x could be caused by a failure of these Ag-bearing DC to migrate to the dLN, or by their decreased survival in secondary lymphoid tissues. To track the ability of transduced DC to migrate to the dLN and mature, we genetically tagged the DC with -gal by immunizing bcl-xfl/fl mice with pCMV-Cre-CAG-lacZ or pCAG-lacZ alone. At various time points after gene gun delivery, we isolated the dLN and evaluated them for -gal activity by flow cytometry (Fig. 4a). Analysis of the absolute number of CD11c+-gal+ cells in the dLN showed 4-fold lower cell counts at 2.5 days in the pCMV-Cre-CAG-lacZ group (1637.5 ± 677) than the pCAG-lacZ group (7103.4 ± 213). The numbers of CD11c+-gal+ DC in the dLN of pCMV-Cre-CAG-lacZ-immunized bcl-xfl/fl mice remained markedly reduced at all time points analyzed, as compared with pCAG-lacZ-immunized mice.
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-gal-marked DC were still detected, albeit in lower numbers, in the dLN of mice immunized with pCMV-Cre-CAG-lacZ, we wished to determine whether increased apoptosis was responsible for their diminished survival. To this end, we analyzed dLN cells for annexin V, a marker of apoptosis. At all time points evaluated, the frequency of CD11c+-gal+annexin V+ cells was greater in the pCMV-Cre-CAG-lacZ group than in the pCAG-lacZ group (Fig. 4b). Thus, the loss of the bcl-x gene resulted in greatly diminished numbers of Ag-bearing DC in the dLN and a concomitant rise in levels of apoptosis in this cell population.
The decreased frequency of marked cells in the dLN could be a consequence of a failure of bcl-x-deficient DC to undergo maturation. Mature DC up-regulate their expression of MHC and T cell costimulatory molecules (2). We analyzed the CD11c+-gal+ population for the expression of I-Ab and CD86, and found no difference after delivery of pCMV-Cre-CAG-lacZ vs pCAG-lacZ (Fig. 4c). From these results, we concluded that the blunted immune responses induced by bcl-x-deficient DC were due to diminished survival of Ag-bearing DC in the dLN, and not due to a maturation block.
bcl-xL isoform is predominant in CD11c+ DC
Multiple isoforms of the bcl-x gene have been described to date, chief among them being the antiapoptotic bcl-xL and the proapoptotic bcl-xS. Because the loxP sites engineered into the genome of the bcl-xfl/fl mouse flank all exons of the gene, cre-mediated deletion results in the loss of all bcl-x isoforms. Results thus far support the hypothesis that sensitivity to apoptosis, stemming from the loss of bcl-xL, results in the diminished frequency of marked cells in the dLN and decreased immune responses. However, an alternative scenario could be envisioned where the presence of bcl-xS can act as a regulator of antiapoptotic molecules including bcl-xL and bcl-2.
To determine the relative abundance of each bcl-x isoform in DC, we designed a primer set to flank the region of bcl-x exon 2 missing in bcl-xS due to alternative RNA splicing; amplification of bcl-xL mRNA would generate a 343-bp fragment (Fig. 5, lane 2), whereas the bcl-xS message would result in a 154-bp fragment (lane 3). We isolated CD11c+ cells from the superficial inguinal lymph nodes of immunologically naive C57BL/6 mice and subjected their mRNA to RT-PCR analysis. The reaction only amplified a 343-bp fragment, suggesting that CD11c+ DC in the inguinal LN predominantly express the bcl-xL isoform. Therefore, the effects seen after deletion of the bcl-x gene in CD11c+ DC would be due to the loss of the antiapoptotic bcl-xL isoform.
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Although the data in Fig. 5 indicate that bcl-xL is the predominant isoform in DC in the lymph node, bcl-xS could still be present at levels undetectable by standard RT-PCR, or it could be critical in regulating immature DC survival in the periphery. Therefore, we sought to specifically silence the bcl-xL isoform in skin-resident DC using siRNA oligonucleotides. DNA vectors based on the pSilencer backbone (Ambion) were engineered to express RNA hairpin oligonucleotides complementary to 21-nt targets. We directed the siRNA oligonucleotides against the 189-bp region of bcl-x exon 2 that is alternatively spliced from bcl-xS mRNA, and tested them for their ability to specifically reduce expression of bcl-xL by transient cotransfection with bcl-xL and bcl-xS expression constructs in human 293 cells. One of two selected siRNAs consistently reduced bcl-xL message by >96%, whereas levels of bcl-xS were unaffected (Fig. 6a). We cloned this siRNA hairpin molecule, along with the RNA Polymerase III U6 promoter, into the pCAG-lacZ vector (pCAG-lacZ-bcl-xL-siRNA). As a control, we similarly produced DNA vaccines containing siRNA oligos targeted against GFP (pCAG-lacZ-GFP-siRNA).
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-gal-marked DC at day 4.5. The frequency of -gal-marked CD11c+ cells in the dLN decreased 4-fold in the pCAG-lacZ-bcl-xL-siRNA-immunized group in comparison with the pCAG-lacZ-immunized group (Fig. 6b). In contrast, the pCAG-lacZ-GFP-siRNA-immunized animals exhibited a similar amount of -gal marking as the positive control (pCAG-lacZ), indicating that effects seen by silencing with the bcl-xL-siRNA are not due to a generalized IFN response to the presence of the siRNA (28).
Posttranscriptional silencing of bcl-xL also resulted in a significant increase in the frequency of -gal-marked cells undergoing apoptosis in the dLN. At 2.5 days postimmunization, there was a 15.2 ± 3.1% increase in the number of CD11c+-gal+annexin V+ cells in the pCAG-lacZ-bcl-xL-siRNA-immunized group over baseline levels in the pCAG-lacZ-immunized group (Fig. 6c). This percentage rapidly increased such that, by day 6.5, the bcl-xL-silenced CD11c+-gal+ DC population had a 6-fold higher frequency of apoptotic cells. By contrast, the untransfected CD11c+-gal– DC population in the pCAG-lacZ-bcl-xL-siRNA-immunized group exhibited similar levels of annexin V+ cells as controls. This signified that the poor rate of survival was unique to the directly transfected DC that had migrated from the site of immunization, and not due to a systemic phenomenon. These results are in full agreement with the data obtained from cre-mediated bcl-x excision in DC of bcl-xfl/fl mice, indicating that it was the loss of the antiapoptotic bcl-xL isoform that produced the survival defect in bcl-x-deficient, migrating CD11c+ DC.
Migration of bcl-xL-deficient DC is not impaired
The data presented in Figs. 4 and 6 suggest that the inability to elicit Ag-specific immune responses is due to the heightened sensitivity of bcl-x-deficient DC to apoptosis. However, it is also possible that the low numbers of CD11c+-gal+ cells in the dLN may also be due to a defect in migration from the skin. To address this issue, we analyzed bcl-xL-deficient DC at the immunization site. We chose to immunize the ears, because isolation of DC from ear skin as well as from the draining auricular lymph nodes is highly tractable. Briefly, we gene gun immunized cohorts of C57BL/6 mice with pCAG-lacZ, pCAG-lacZ-bcl-xL-siRNA, or pCAG-lacZ-GFP-siRNA. At 1.5 and 2.5 days after vaccination, we quantified the absolute number of CD11c+-gal+ cells that remained at the immunization site or that migrated to the dLN. There was no significant difference among the three groups in the absolute numbers of Ag-bearing cells per ear at either time point. Although 1000 CD11c+-gal+ cells were still present at the immunization site at day 1.5, only 250 remained by day 2.5 (Fig. 7a). We also analyzed the DC at the immunization site for evidence of apoptosis by annexin V staining. We found no difference among the three groups in levels of annexin V in the CD11c+-gal+ population isolated from the ear at both time points (data not shown), suggesting that DC were not dying at the immunization site, but had migrated away from the skin.
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-gal+ cells, we analyzed the draining auricular lymph nodes for the presence of Ag-bearing DC. Similar to the results observed with immunization of the abdominal skin, we found reduced numbers of bcl-xL-deficient, CD11c+-gal+ DC in the dLN at both time points (Fig. 7b). In comparison, we enumerated 500 and 1500 CD11c+-gal+ DC per dLN at days 1.5 and 2.5, respectively, from mice immunized with pCAG-lacZ or pCAG-lacZ-GFP-siRNA. This increase in CD11c+-gal+ DC in the dLN mirrored a concomitant decrease in CD11c+-gal+ DC at the immunization site of both control groups. The corresponding bcl-xL-deficient population showed a similar reduction in numbers of Ag-bearing DC at the immunization site at day 2.5; however, there were persistently low numbers of CD11c+-gal+ cells in the dLN, due to higher levels of apoptosis. Taken together, these data indicate that bcl-xL-deficient DC are capable of migration from the periphery, and support the hypothesis that impaired longevity of the Ag-bearing DC in the dLN causes the failure to elicit immune responses. Delivery of bcl-xL in trans complements bcl-x deficiency
To further confirm that the bcl-xL isoform is indeed critical for mature DC survival, we attempted to rescue the phenotype observed with bcl-x-deficient DC by providing bcl-xL in trans. bcl-xfl/fl mice were immunized with gene gun bullets that had been cocoated with the pCMV-Cre-CAG-lacZ DNA vector, along with a second plasmid encoding either bcl-xL cDNA, or a mutant bcl-xL (bcl-xL mt7) modified in the BH1 domain to abrogate its antiapoptotic function (29). At 4.5 days after immunization, we evaluated CD11c+ cells from the dLN for their expression of -gal and annexin V. Complementation of cre-mediated, bcl-x deficiency with wild-type bcl-xL, but not bcl-xL mt7, restored the numbers of -gal-marked CD11c+ DC in the dLN (Fig. 8a). Furthermore, the addition of wild-type bcl-xL was sufficient to protect the transfected DC from apoptotic death, showing a 21.55 ± 6.8% reduction in the percentage of annexin V+ cells in the CD11c+-gal+ population (Fig. 8b). However, complementation with the mutant bcl-xL did not produce any significant change in the level of apoptotic cells. Thus, rescue of bcl-x deficiency in CD11c+ DC was accomplished by delivery of wild-type bcl-xL in trans.
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Discussion |
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-gal into a conditional bcl-x knockout mouse (bcl-xfl/fl) can render skin-resident DC deficient in bcl-x, and mark them with -gal. Using this strategy, we showed that bcl-x-deficient DC were capable of maturation and migration to dLN. However, their numbers were progressively decreased due to apoptosis, signifying that this gene is involved in the survival of mature DC. The lack of immunostimulation is thus not due to an intrinsic defect in the DC, but is instead due to the failure to maintain adequate numbers of Ag-bearing APC in the dLN. In addition, the concordance of the effects seen with siRNA-mediated bcl-xL silencing with the results of cre-mediated bcl-x excision in the bcl-xfl/fl mouse indicates that bcl-xL plays a pivotal role in maintaining the longevity of Ag-bearing DC after migration to the dLN. Our data supplement findings that overexpression of antiapoptotic factors such as bcl-2 and bcl-xL in vivo enhances mature DC longevity in secondary lymphoid tissues, and correspondingly increases B and T cell responses (30, 31). In DC, TLR signaling induced by inflammatory stimuli has been shown to up-regulate expression of both bcl-2 and bcl-xL, and other antiapoptotic proteins (33). Furthermore, the addition of T cell-derived factors, such as TRANCE and CD40L, to DC cultures can also promote their survival, thus implicating the DC-T cell interaction in the cellular homeostasis of Ag-bearing DC (10, 11, 32). TRANCE ligation inhibited apoptosis by selective up-regulation of bcl-xL expression, and concomitantly improved the T cell stimulatory capacity of DC (34). In contrast, CD40 signaling increased levels of the antiapoptotic protein bcl-2, and has also been shown to prolong the longevity and Ag-presenting capacity of DC (12, 32). Although it is possible that both bcl-xL and bcl-2 fulfill redundant roles in protection from cell death, gene microarray studies detected a significant increase in bcl-xL expression immediately after DC maturation, whereas bcl-2 remained at basal levels (13). Additionally, Kim et al. (29) found that Ag-specific immunostimulation by DNA vaccines was most optimally enhanced by engineering constructs to coexpress bcl-xL, rather than other antiapoptotic proteins such as bcl-2. Indeed, our studies show that bcl-xL-deficient DC are unable to maintain their survival in the dLN, implying that the loss of this antiapoptotic gene is sufficient to drive these cells to an accelerated death.
Recent reports (35, 36, 37) have suggested that the initiation of T cell responses may not require prolonged interaction with Ag and APC. This implies that DC longevity may not play such a crucial role in the initiation of T cell responses, and that, after the initial interaction between T cell and DC, the T cell may be able to autonomously differentiate and exert effector functions. However, our studies seem to suggest that survival of Ag-bearing DC is critical for the generation of immune responses. bcl-x-deficient DC were still capable of migration from the immunization site, although they were present in the dLN at much fewer numbers due to higher levels of apoptosis. At the peak of migration (2.5 days after gene gun immunization) (3), there were only 1600 Ag-bearing skin-derived DC per draining superficial inguinal lymph node. Indeed, not only were the frequencies of marked cells in the dLN consistently lower, the rate of decline of the bcl-x-deficient DC population was much more rapid than that seen in the mice immunized with the tracking Ag alone. By day 6.5, the absolute number of CD11c+-gal+ cells had dropped to 250 per lymph node, whereas >5000 could be detected in the control group. The factthat immune responses were so markedly diminished by the decrease in the number of -gal-marked, bcl-x-deficient DC in the dLN suggests that there may be a threshold number of Ag-bearing DC required to induce acquired immunity. This may be due to the inability of the few DC to find the small number of Ag-specific T cells. Recent work by our laboratory has shown that previous reports enumerating the peak frequencies of Ag-bearing DC had underestimated the actual numbers by 100-fold (3), which raises the possibility that optimal immune responses require a higher number of DC than was achieved in the environment of bcl-x deficiency.
The results presented in this study have implications for the rational design of DC-based vaccine regimens. As vaccine-mediated immunity is thought to rely upon APC, especially DC, it is important to find ways by which DC-driven Ag-specific responses can be heightened. Previous studies have attempted to ameliorate the level of immune responses by inducing apoptosis in the Ag-bearing cell (38, 39, 40), such that many other APC can take up the apoptotic body and initiate cellular immunity to a greater degree than could the small number of DC targeted by the vaccine. However, shortening the life span of Ag-bearing DC in the dLN by targeted bcl-xL deficiency did not improve, but instead abrogated immune responses. Our work suggests that it may be more critical to up-regulate the expression of antiapoptotic molecules like bcl-xL in the APC. Increasing the survival of the stimulatory APC in the secondary lymphoid organs could represent a major step in improving the strength of Ag-specific immunity.
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Acknowledgments |
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Footnotes |
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1 This work was supported by a grant to J.J. from the National Institutes of Health. 
2 Address correspondence and reprint requests to Dr. Joshy Jacob, Department of Microbiology and Immunology, Emory Vaccine Center, Emory University, 954 Gatewood Road NE, Atlanta, GA 30329. E-mail address: jjacob3{at}emory.edu
3 Abbreviations used in this paper: DC, dendritic cell; dLN, draining lymph node; TRANCE, TNF-related activation-induced cytokine; CAG, chicken -actin; HA, hemagglutinin; siRNA, small interfering RNA; -gal, -galactosidase.
Received for publication April 29, 2004. Accepted for publication July 22, 2004.
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References |
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1 promotes in vitro generation of dendritic cells by protecting progenitor cells from apoptosis. J. Immunol. 158:1591.[Abstract]
maintains the viability of murine epidermal Langerhans cells in culture, but in contrast to granulocyte/macrophage colony-stimulating factor, without inducing their functional maturation. J. Exp. Med. 171:159. geo 26 gene trap strain leads to widespread expression of -galactosidase in mouse embryos and hematopoietic cells. Proc. Natl. Acad. Sci. USA 94:3789.-D-galactosidase activity after transduction of Escherichia coli lacZ. Proc. Natl. Acad. Sci. USA 85:2603.Related articles in The JI:
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 B. K. Choi, Y. H. Kim, P. M. Kwon, S. C. Lee, S. W. Kang, M. S. Kim, M. J. Lee, and B. S. Kwon 4-1BB Functions As a Survival Factor in Dendritic Cells J. Immunol., April 1, 2009; 182(7): 4107 - 4115. [Abstract] [Full Text] [PDF]
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 C. Jakubzick, M. Bogunovic, A. J. Bonito, E. L. Kuan, M. Merad, and G. J. Randolph Lymph-migrating, tissue-derived dendritic cells are minor constituents within steady-state lymph nodes J. Exp. Med., November 24, 2008; 205(12): 2839 - 2850. [Abstract] [Full Text] [PDF]
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 Y. Oshima, T. Akiyama, A. Hikita, M. Iwasawa, Y. Nagase, M. Nakamura, H. Wakeyama, N. Kawamura, T. Ikeda, U.-i. Chung, et al. Pivotal Role of Bcl-2 Family Proteins in the Regulation of Chondrocyte Apoptosis J. Biol. Chem., September 26, 2008; 283(39): 26499 - 26508. [Abstract] [Full Text] [PDF]
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 A. Varas, C. Hernandez-Lopez, J. Valencia, S. Mattavelli, V. G. Martinez, L. Hidalgo, C. Gutierrez-Frias, A. G. Zapata, R. Sacedon, and A. Vicente Survival and function of human thymic dendritic cells are dependent on autocrine Hedgehog signaling J. Leukoc. Biol., June 1, 2008; 83(6): 1476 - 1483. [Abstract] [Full Text] [PDF]
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 H. Li, Y. Chen, A. F. Jones, R. H. Sanger, L. P. Collis, R. Flannery, E. C. McNay, T. Yu, R. Schwarzenbacher, B. Bossy, et al. Bcl-xL induces Drp1-dependent synapse formation in cultured hippocampal neurons PNAS, February 12, 2008; 105(6): 2169 - 2174. [Abstract] [Full Text] [PDF]
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L. Gutierrez, T. Nikolic, T. B. van Dijk, H. Hammad, N. Vos, M. Willart, F. Grosveld, S. Philipsen, and B. N. Lambrecht Gata1 regulates dendritic-cell development and survival Blood, September 15, 2007; 110(6): 1933 - 1941. [Abstract] [Full Text] [PDF]
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T. Izawa, N. Ishimaru, K. Moriyama, M. Kohashi, R. Arakaki, and Y. Hayashi Crosstalk between RANKL and Fas signaling in dendritic cells controls immune tolerance Blood, July 1, 2007; 110(1): 242 - 250. [Abstract] [Full Text] [PDF]
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W. L. W. Chang, N. Baumgarth, M. K. Eberhardt, C. Y. D. Lee, C. A. Baron, J. P. Gregg, and P. A. Barry Exposure of Myeloid Dendritic Cells to Exogenous or Endogenous IL-10 during Maturation Determines Their Longevity J. Immunol., June 15, 2007; 178(12): 7794 - 7804. [Abstract] [Full Text] [PDF]
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S. Pradhan, J. Genebriera, W. L. Denning, K. Felix, C. A. Elmets, and L. Timares CD4 T Cell-Induced, Bid-Dependent Apoptosis of Cutaneous Dendritic Cells Regulates T Cell Expansion and Immune Responses J. Immunol., November 1, 2006; 177(9): 5956 - 5967. [Abstract] [Full Text] [PDF]
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D. Xia, S. Hao, and J. Xiang CD8+ Cytotoxic T-APC Stimulate Central Memory CD8+ T Cell Responses via Acquired Peptide-MHC Class I Complexes and CD80 Costimulation, and IL-2 Secretion. J. Immunol., September 1, 2006; 177(5): 2976 - 2984. [Abstract] [Full Text] [PDF]
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 O. Shefi, C. Simonnet, M. W. Baker, J. R. Glass, E. R. Macagno, and A. Groisman Microtargeted gene silencing and ectopic expression in live embryos using biolistic delivery with a pneumatic capillary gun. J. Neurosci., June 7, 2006; 26(23): 6119 - 6123. [Full Text] [PDF]
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J. Yang, S. P. Huck, R. S. McHugh, I. F. Hermans, and F. Ronchese Perforin-dependent elimination of dendritic cells regulates the expansion of antigen-specific CD8+ T cells in vivo PNAS, January 3, 2006; 103(1): 147 - 152. [Abstract] [Full Text] [PDF]
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 C. H. Schimmelpfennig, S. Schulz, C. Arber, J. Baker, I. Tarner, J. McBride, C. H. Contag, and R. S. Negrin Ex Vivo Expanded Dendritic Cells Home to T-Cell Zones of Lymphoid Organs and Survive in Vivo after Allogeneic Bone Marrow Transplantation Am. J. Pathol., November 1, 2005; 167(5): 1321 - 1331. [Abstract] [Full Text] [PDF]
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J. M. Savitt, S. S. Jang, W. Mu, V. L. Dawson, and T. M. Dawson Bcl-x Is Required for Proper Development of the Mouse Substantia Nigra J. Neurosci., July 20, 2005; 25(29): 6721 - 6728. [Abstract] [Full Text] [PDF]
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N. Zhang and Y.-W. He The Antiapoptotic Protein Bcl-xL Is Dispensable for the Development of Effector and Memory T Lymphocytes J. Immunol., June 1, 2005; 174(11): 6967 - 6973. [Abstract] [Full Text] [PDF]
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