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  1. 1. The Journal of Immunology bcl-xL Is Critical for Dendritic Cell Survival In Vivo1 Huiming Hon,* Edmund B. Rucker III,† Lothar Hennighausen,‡ and Joshy Jacob2* Dendritic cells (DC) are important regulators of immune function, transporting Ags from the periphery to draining lymph nodes (dLN) where they prime Ag-specific T lymphocytes. The magnitude of the immune response generated depends upon the longevity of the Ag-bearing DC in lymphoid tissues. We hypothesized that the control of DC survival is regulated by the antiapoptotic factor bcl-xL. Gene gun immunization of dual-expression DNA vaccines into a bcl-xfl/fl mouse resulted in the delivery of Ag, as well as selective deletion of the bcl-x gene in directly transfected, skin-residing DC. bcl-x-deficient DC failed to mount effective immune responses, and this corresponded to their rapid disappearance from the dLN due to apoptosis. We confirmed these results using RNA interference to specifically silence the antiapoptotic bcl-xL isoform in targeted skin-residing DC of C57BL/6 mice. In addition, delivery of bcl-xL in trans complemented the bcl-x deficiency in DC of bcl-xfl/fl mice, resulting in the maintenance of normal levels of Ag-bearing DC in the dLN. Taken together, our work demonstrates that the bcl-xL isoform is critical for survival of skin- derived, Ag-bearing DC in vivo. The Journal of Immunology, 2004, 173: 4425– 4432. D endritic cells (DC)3 are key regulators of the immune mRNA levels of another antiapoptotic protein, bcl-2, decreased as system. They exist in an immature form in the periphery, early as 4 h after activation. Collectively, these data suggest that constantly sampling the tissue microenvironment (1). the maintenance of mature DC survival is likely to involve bcl-x Upon microbial insult, DC process Ag and differentiate into pro- genes; however, it has yet to be tested in vivo. fessional APC. These mature DC secrete cytokines and have in- The bcl-x gene encodes multiple isoforms, including antiapop- creased cell surface expression of both Ag-MHC complexes and T totic bcl-xL and proapoptotic bcl-xS (14). bcl-xL localizes to the cell costimulatory molecules (2). Due to a differential expression mitochondrial membrane, where it antagonizes the ability of bax or of chemokine receptors, they migrate to draining lymph nodes bak to release cytochrome c from the mitochondrion and induce a (dLN) where these Ag-bearing DC initiate specific adaptive im- caspase-mediated cascade leading to cell death (15). Unlike the mune responses. We showed previously that the migration of ma- antiapoptotic bcl-xL, the proapoptotic bcl-x isoform bcl-xS lacks ture DC to the dLN peaks at 2.5 days after a stimulus, with Ag- the C-terminal 189 bp of exon 2 due to the use of an alternative bearing DC detectable in the dLN for up to 14 days (3). This period splice site (16). bcl-x knockout mice die at embryonic day 13, with can determine the extent of T cell priming, and thus plays a role in signs of apoptosis in immature neurons of the brain, and in hemo- regulating the strength of the immune response. The control of DC poietic cells in the liver (17), prompting the generation of cre-lox longevity in secondary lymphoid tissues is probably controlled by conditional bcl-x knockout mice (bcl-xfl/fl) (18, 19). the interplay between T cell signals, secreted cytokines, and up- To evaluate the role of bcl-xL in DC function, we used biolistic regulation of antiapoptotic factors. delivery of cre recombinase-encoding DNA vaccines to selectively Isolated DC are prone to spontaneous apoptosis, and they can be knock out the bcl-x gene in skin-resident DC of bcl-xfl/fl mice. In rescued by factors such as GM-CSF, TNF- , or TGF- 1 (4 –7), conjunction, we also used RNA interference technology to specif- which induce antiapoptotic pathways mediated by bcl-2 and bcl-xL ically silence the bcl-xL isoform in DC. Our studies show that the (8, 9). Furthermore, the addition of TNF-related activation-induced antiapoptotic isoform bcl-xL is critical for the survival of Ag-bear- cytokine (TRANCE) and CD40L to cultures improves the survival ing DC in secondary lymphoid tissues. bcl-xL-deficient DC under- of DC, correlating with an increased expression of bcl-xL and went extensive apoptosis, and failed to induce protective immune bcl-2, respectively (10 –12). Analysis of the gene expression pro- responses in vivo. file 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, Materials and Methods Mice *Department of Microbiology and Immunology, Emory Vaccine Center, Emory Uni- versity, Atlanta, GA 30329; †Department of Animal Sciences, University of Missouri, bcl-xfl/fl mice were imported from the University of Missouri, and analyzed Columbia, MO 65211; and ‡Laboratory of Genetics and Physiology, National Insti- for genotype by PCR as previously described (18, 19). C57BL/6 mice were tute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, obtained from Charles River Labs (Wilmington, MA). Mice used for ex- Bethesda, MD 20892 periments were between 4 and 10 wk of age, and maintained under stan- Received for publication April 29, 2004. Accepted for publication July 22, 2004. dard pathogen-free housing conditions at the Emory Vaccine Center viva- rium with the approval of the Institutional Animal Care and Use The costs of publication of this article were defrayed in part by the payment of page Committee at Emory University. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a grant to J.J. from the National Institutes of Health. PCR and RT-PCR analyses 2 Address correspondence and reprint requests to Dr. Joshy Jacob, Department of Microbiology and Immunology, Emory Vaccine Center, Emory University, 954 Gate- PCR identification of the recombined bcl-x locus in bcl-xfl/fl mice was per- wood Road NE, Atlanta, GA 30329. E-mail address: jjacob3@emory.edu formed as previously described (18, 19). To detect bcl-xL and bcl-xS iso- 3 Abbreviations used in this paper: DC, dendritic cell; dLN, draining lymph node; forms, primers flanking the C-terminal 189 bp of exon 2 were used in a TRANCE, TNF-related activation-induced cytokine; CAG, chicken -actin; HA, standard RT-PCR: forward primer, 5 -GGAGAGCGTTCAGTGATC-3 , hemagglutinin; siRNA, small interfering RNA; -gal, -galactosidase. and reverse primer, 5 -CCAGCCACAGTCATGCC-3 . Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00
  2. 2. 4426 bcl-xL IS CRITICAL FOR DC SURVIVAL IN VIVO DNA vaccine plasmids Epidermal cell preparation Cre recombinase cDNA coupled with a SV40-derived nuclear localization To isolate epidermal cell suspensions, we excised the immunized ears at sequence was cloned downstream of the CMV immediate-early promoter the base, and split-thickness ears were treated with 0.5% trypsin for 30 min and upstream of a bovine growth hormone polyadenylation sequence at 37°C. Following incubation, a single-cell suspension was made by dis- (pCMV-Cre). Similarly, plasmids expressing full-length OVA cDNA rupting epidermal sheets in a 70- m cell strainer. (pCMV-OVA) and influenza hemagglutinin (HA) cDNA (pCMV-HA) were constructed. To track Ag-bearing cells, the lacZ gene was cloned Flow cytometry downstream of the chicken -actin (CAG) promoter (pCAG-lacZ). To gen- erate dual-expression DNA vaccines, these Ag expression cassettes were For flow cytometric analysis of -galactosidase ( -gal) expression, colla- excised and blunt-end ligated into the cre-expressing vector. Dual-expres- genase-treated lymph node cells or epidermal cell suspensions from ear sion bcl-xL small interfering RNA (siRNA) plasmids were assembled by skin were hypotonically loaded with 0.5 mM fluorescein di-D-galactopyr- cloning the U6-bcl-xL-siRNA constructs downstream of pCAG-lacZ. Plas- anoside (Molecular Probes, Eugene, OR), as previously described (23–25). mids encoding pBcl-xS, pBcl-xL, and the mt7 pBcl-xL mutant were kind Aliquots of fluorescein di-D-galactopyranoside-loaded cells were stained gifts from T. W. Behrens (University of Minnesota, Minneapolis, MN), S. with fluorochrome-conjugated Abs (anti-CD11c-allophycocyanin, -CD86- Korsmeyer (Harvard University, Boston, MA), and J. M. Hardwick (Johns biotin, -I-Ab-PE, -annexin V-PE; BD Biosciences), with a secondary streptavidin-PerCP (BD Biosciences) Ab staining when needed. Cells were Hopkins University, Baltimore, MD), respectively. then acquired without fixation with a FACSCalibur (BD Biosciences), and data were analyzed with FlowJo software (Tree Star, Ashland, OR). We Immunizations sorted cells to 94% purity with a FACSVantage (BD Biosciences), iso- Gene gun immunizations were performed on shaved abdominal skin of lating CD11c cells from superficial inguinal lymph nodes of a naive mice as described previously (3, 20). Briefly, a hand-held Helios gene C57BL/6 mouse, as well as CD11c -gal and CD11c -gal popula- delivery system (Bio-Rad, Hercules, CA) was used to deliver two doses of tions from MACS-enriched CD11c cells from dLN of bcl-xfl/fl mice im- 0.5 g of plasmid DNA coated on 1 mg of gold beads (DeGussa-Huls, munized 2.5 days earlier with pCMV-Cre-CAG-lacZ. Ridgefield Park, NJ) at a helium pressure of 400 psi. In experiments de- signed to study DC at the immunization site, we immunized ears of mice Statistical analyses with one shot each on the dorsal and ventral sides (0.5 g of plasmid DNA Statistical differences between experimental groups were analyzed with per shot) using the Helios gene gun set at a helium pressure of 600 psi. Prism software (GraphPad, San Diego, CA), with values of p 0.05 from Student’s t test considered significant. T cell proliferation Mice were immunized with pCMV-OVA, pCMV-Cre-CMV-OVA, or Results empty DNA vector, and draining superficial inguinal lymph nodes were Targeted deletion of bcl-x in DC removed and pooled 5 days later. CD11c cells were enriched to 90% purity by anti-CD11c (N418)-coupled magnetic beads (Miltenyi Biotec, Previously, we had permanently marked and tracked the migration Auburn, CA). Responder T cells were isolated from spleens of OT-II TCR of Ag-bearing DC from the periphery to dLN by biolistic delivery transgenic mice by incubation of the single-cell suspension with anti-CD3- of cre-encoding plasmids into a cre-indicator mouse strain, FITC (eBiosciences, San Diego, CA) Ab, followed by positive selection ROSA26R (3). We showed that DC resident in the skin could be with anti-FITC microbeads (Miltenyi Biotec). CD11c DC from immu- directly transduced in vivo via gene gun immunization. In this nized 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 study, we used the same approach to permanently delete the bcl-x (OVA257–264) and OT-II (OVA323–339) peptides were added to wells with gene from the genome of cells in bcl-xfl/fl mice. These mice have OT-II T cells. Cells were pulsed with 1 Ci of [3H]thymidine after 48 h of been engineered with two loxP sites flanking the bcl-x gene; in the culture, and assayed for radionucleotide incorporation in a MicroBeta plate presence of bacteriophage P1-derived cre recombinase, homolo- reader (Wallac, Turku, Finland) the following day. gous recombination occurs between these two loxP sites, resulting Serum ELISA 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 At days 15 and 30 after gene gun immunization, serum samples were DNA vaccine encoding cre recombinase (pCMV-Cre). The immu- collected from 50 l of blood obtained from mice via retro-orbital bleeds, and assayed on ELISA plates coated with sucrose gradient-purified nization site was marked, and then excised 6 h later and subjected A/PR/8/34 (H1N1) influenza virus. Quantitation of Ab levels was per- to genomic DNA extraction. As controls, the same mouse received formed by comparison with a standard curve using goat anti-mouse IgG immunizations at nonoverlapping sites with either pCMV-OVA or (BD Biosciences, San Jose, CA), and data were analyzed with Microplate gold particles alone. We then analyzed the genomic DNA samples Reader software (Bio-Rad). for cre-mediated deletion of the bcl-x gene by PCR, using a primer Influenza challenge set that produces a 150-bp fragment only from a deleted bcl-x locus (18, 19). Amplification of the 150-bp recombination product Forty days following DNA vaccination with constructs expressing influ- was restricted to the area of skin that had been immunized with cre enza HA, animals were challenged with a mouse-adapted influenza virus A/PR/8/34 (H1N1), a kind gift from H. Robinson (Emory University, At- recombinase (Fig. 1a). The adjacent areas of skin that were im- lanta, GA). Allantoic fluid containing influenza virus was serially diluted in munized with pCMV-OVA or empty gold particles did not pro- sterile PBS with 0.2% BSA (Sigma-Aldrich, St. Louis, MO), and a 50- l duce the 150-bp recombination product, indicating that we could volume containing 3 LD50 (0.03 HAU) viral dose was given intranasally achieve a targeted, cre-dependent deletion of the bcl-x gene. The to anesthetized mice. Following viral challenge, total body weight and presence of two copies of the undeleted bcl-x gene was verified by survival were recorded over the subsequent 10 days. PCR in all control DNA samples (data not shown). siRNA constructs and testing To confirm cre-mediated deletion of the bcl-x gene in trans- fected DC, we immunized cohorts of bcl-xfl/fl mice with DNA vac- Candidate siRNA oligos targeted against the 189-bp region at the 3 end of cines dually encoding cre recombinase and -gal. In these vectors, exon 2 of murine bcl-x were devised according to guidelines established by Tuschl (21). The siRNA oligos were synthesized and cloned into the pSi- the strong hybrid CMV enhancer and CAG promoter drove ex- lencer 1.0 vector under control of the U6 RNA Polymerase III promoter per pression of -gal to permit tracking of the small percentage of manufacturer’s instructions (Ambion, Austin, TX). Of the two siRNA con- transfected cells. At 2.5 days after immunization, both the structs tested, the greatest knockdown was seen with the oligo targeting CD11c -gal and the CD11c -gal populations were sorted bcl-x468 – 488 (5 -AAGGAGATGCAGGTATTGGTG-3 ). Ability to silence bcl-xL was tested by transient cotransfection with expression vectors en- from draining superficial inguinal nodes, and subjected to genomic coding murine bcl-xL or bcl-xS into human 293 cells. The control GFP DNA extraction and PCR amplification for the 150-bp recombi- siRNA oligo was constructed following Sui et al. (22). nation product. The CD11c -gal , but not the CD11c -gal
  3. 3. The Journal of Immunology 4427 alone (2.05 0.1 vs 18.32 0.6 103 cpm, respectively). Re- sponder T cells pulsed with cognate OT-II peptide showed maxi- mal amounts of proliferation (25.45 2 103 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 FIGURE 1. Specific deletion of bcl-x gene in bcl-xfl/fl mice following resulted in a drastic loss of their T cell-priming capacity. gene gun immunization with cre recombinase. a, Mice (n 3) were gene gun immunized with nonoverlapping shots of pCMV-Cre (site 1), pCMV- bcl-x-deficient DC elicit reduced Ab responses OVA (site 2), or empty bullets (site 3). The immunization sites were To determine whether humoral immune responses would be marked and excised 6 h later. Genomic DNA isolated from excised skin blunted by the loss of bcl-x in Ag-bearing DC, we used the mouse sections at each site and from the tail was subjected to PCR analysis for influenza infection model system. Protection from influenza infec- cre-mediated bcl-x deletion. Amplification of the 150-bp recombination product occurred only with DNA from the area immunized with pCMV- tion depends upon the generation of Abs against the viral HA Cre. Amplification of -actin was used as a PCR positive control. Data protein (26). DNA vaccines encoding HA induce long-lasting and from one mouse are shown; comparative data were obtained in all exper- high titers of specific Abs (27). We immunized groups of bcl-xfl/fl iments. b, Mice (n 5) were immunized with pCAG-lacZ-CMV-Cre, and mice with a DNA vaccine coexpressing cre recombinase and in- dLN were pooled at 2.5 days. The CD11c -gal and CD11c -gal fluenza HA (pCMV-Cre-CMV-HA), or with controls pCMV-HA populations were isolated by cell sorting, and subjected to genomic DNA or empty vector. At days 15 and 30 postimmunization, we col- extraction. PCR analysis showed the bcl-x recombination product in the lected sera from each mouse and measured the concentration of CD11c -gal (lane 1), but not in the CD11c -gal (lane 2) population. anti-HA Abs by a standard ELISA (Fig. 3a). Mice immunized with pCMV-HA exhibited 24.37 2.4 g/ml anti-HA IgG at day 15, and 77.39 7.4 g/ml at day 30 after vaccination. In contrast, population exhibited the 150-bp recombination product (Fig. 1b), mice immunized with pCMV-HA-CMV-Cre produced signifi- indicating that biolistic delivery of the dual-expression plasmid cantly lower levels of influenza-specific Abs at both time points deleted the bcl-x gene in skin-residing DC, which could then be (7.23 2.6 and 9.3 3 g/ml; p 0.05), whereas the response tracked to the dLN. of control mice immunized with an empty DNA vector remained at baseline levels. 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 im- munized 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-spe- cific T cell proliferation, yielding a lower level of [3H]thymidine uptake than that achieved from immunization with pCMV-OVA FIGURE 3. Cre-mediated excision of the bcl-x gene in DC leads to FIGURE 2. Reduced T cell stimulatory capacity of CD11c DC after lowered humoral responses. a, Cohorts (n 5) of bcl-xfl/fl mice were im- loss of the bcl-x gene. Groups of bcl-xfl/fl mice (n 5) were immunized munized with pCMV-Cre-CMV-HA, pCMV-HA, or an empty vector. Se- with pCMV-Cre-CMV-OVA, pCMV-OVA, pCMV-Cre, or an empty vec- rum Ab levels of anti-HA IgG at 15 and 30 days after gene gun immuni- tor. CD11c DC isolated from dLN 5 days later were pooled and used zation were quantified by ELISA for each individual mouse. The mean IgG directly in vitro, without exogenous peptide, to stimulate proliferation of concentrations ( SEM) are plotted. b, The same cohorts of mice were naive, OVA-specific, OT-II TCR transgenic T cells. In control wells, re- challenged intranasally with 3 LD50 of mouse-adapted influenza virus sponder T cells were pulsed with OT-I and OT-II peptides for negative and A/PR/8/34 at day 40 after immunization, and monitored for weight loss positive controls, respectively. Results are shown as the mean [3H]thymidine over the subsequent 10 days. Mean percent baseline weight ( SEM) at day uptake of duplicate wells after 72-h culture; error bars indicate range from the 5, the peak of viral infection, is graphed. Comparable data were obtained mean. Comparable data were obtained in two separate experiments. in two separate experiments. , p 0.05.
  4. 4. 4428 bcl-xL IS CRITICAL FOR DC SURVIVAL IN VIVO We then infected the same groups of mice with 3 LD50 of a CD11c -gal cells in the dLN showed 4-fold lower cell mouse-adapted influenza strain A/PR/8/34 at day 40 after immu- counts at 2.5 days in the pCMV-Cre-CAG-lacZ group nization, and monitored them daily for weight loss (Fig. 3b). At the (1637.5 677) than the pCAG-lacZ group (7103.4 213). The peak of infection (day 5), the pCMV-HA-CMV-Cre-immunized numbers of CD11c -gal DC in the dLN of pCMV-Cre- mice had dropped to an average of 79.8 1.2% of their baseline CAG-lacZ-immunized bcl-xfl/fl mice remained markedly re- weight, and the control empty vector group weighed in at 78.9 duced at all time points analyzed, as compared with 1.1% of baseline. Both of these cohorts quickly succumbed to the pCAG-lacZ-immunized mice. viral infection. However, mice that received pCMV-HA showed a As -gal-marked DC were still detected, albeit in lower num- higher level of protection from viral challenge, as would be ex- bers, in the dLN of mice immunized with pCMV-Cre-CAG-lacZ, pected from their high anti-HA Ab titers, maintaining 89.7 0.3% we wished to determine whether increased apoptosis was respon- of their baseline weight at day 5 postinfection. The failure of the sible for their diminished survival. To this end, we analyzed dLN bcl-x-deficient DC to induce anti-HA Abs was reflected in the cells for annexin V, a marker of apoptosis. At all time points eval- drastic weight loss of the pCMV-Cre-CMV-HA cohort upon viral uated, the frequency of CD11c -gal annexin V cells was challenge. Thus, Ag-bearing DC that had undergone cre-mediated greater in the pCMV-Cre-CAG-lacZ group than in the pCAG-lacZ deletion of bcl-x demonstrated a diminished capacity to elicit hu- group (Fig. 4b). Thus, the loss of the bcl-x gene resulted in greatly moral immune responses. diminished numbers of Ag-bearing DC in the dLN and a concom- itant rise in levels of apoptosis in this cell population. bcl-x is needed for survival of DC The decreased frequency of marked cells in the dLN could be a The loss of immunostimulatory capacity in DC established by the consequence of a failure of bcl-x-deficient DC to undergo matu- loss of bcl-x could be caused by a failure of these Ag-bearing DC ration. Mature DC up-regulate their expression of MHC and T cell to migrate to the dLN, or by their decreased survival in secondary costimulatory molecules (2). We analyzed the CD11c -gal lymphoid tissues. To track the ability of transduced DC to migrate population for the expression of I-Ab and CD86, and found no to the dLN and mature, we genetically tagged the DC with -gal difference after delivery of pCMV-Cre-CAG-lacZ vs pCAG-lacZ by immunizing bcl-xfl/fl mice with pCMV-Cre-CAG-lacZ or (Fig. 4c). From these results, we concluded that the blunted im- pCAG-lacZ alone. At various time points after gene gun delivery, mune responses induced by bcl-x-deficient DC were due to dimin- we isolated the dLN and evaluated them for -gal activity by ished survival of Ag-bearing DC in the dLN, and not due to a flow cytometry (Fig. 4a). Analysis of the absolute number of maturation block. FIGURE 4. bcl-x deletion in DC leads to normal maturation, but enhanced apoptosis in dLN over time. a, bcl-xfl/fl mice (n 2) were immunized with pCMV-Cre-CAG-lacZ or pCAG-lacZ, and dLN were analyzed by flow cytometry for -gal expression at various time points. Mice immunized with pCMV-Cre-CAG-lacZ had a consistently diminished absolute number of CD11c -gal cells that migrated to the dLN, compared with bcl-xfl/fl mice immunized with pCAG-lacZ alone. Representative data from two independent experiments are shown, and have been gated on the early apoptotic 7-aminoactinomycin D (7AAD)lowCD11c population. b, Enhanced apoptosis in the bcl-x-deficient, -gal population was observed as a higher percentage of annexin V cells. The data are represented as percent change in frequency of CD11c -gal annexin V DC in experimental mice vs pCAG-lacZ- immunized control mice. c, At day 2.5, bcl-x-deficient DC exhibited comparable expression of activation markers, MHC class II I-Ab and CD86, as control cells. The flow cytometry histograms plot the CD11c -gal DC from animals immunized with pCMV-Cre-CAG-lacZ (solid lines) or pCAG-lacZ (dotted lines). Shaded region represents the isotype Ab control.
  5. 5. The Journal of Immunology 4429 the dLN and decreased immune responses. However, an alterna- tive 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 FIGURE 5. CD11c DC express the bcl-xL, but not the bcl-xS isoform. lymph nodes of immunologically naive C57BL/6 mice and sub- Schematic of the murine bcl-x gene. The 189-bp segment that is alterna- jected their mRNA to RT-PCR analysis. The reaction only ampli- tively spliced from the bcl-xS isoform is indicated by crosshatches. Am- fied a 343-bp fragment, suggesting that CD11c DC in the ingui- plification of mRNA using a primer set (small arrows) flanking the segment nal LN predominantly express the bcl-xL isoform. Therefore, the missing in bcl-xS can distinguish between the bcl-xL isoform (343-bp prod- effects seen after deletion of the bcl-x gene in CD11c DC would uct) and the bcl-xS isoform (154-bp product). CD11c DC were isolated by be due to the loss of the antiapoptotic bcl-xL isoform. cell sorting from the superficial inguinal lymph nodes of naive C57BL/6 mice, and subjected to mRNA extraction. RT-PCR analysis reveals the presence of the bcl-xL, but not the bcl-xS isoform (lane 1). As a reference, RNA interference silencing supports the role of bcl-xL in DC RT-PCR analyses of mRNA from human kidney cell line 293 transfected with pBcl-xL (lane 2) or pBcl-xS (lane 3) are shown. Data are representa- survival tive of three separate experiments. 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 bcl-xL isoform is predominant in CD11c DC regulating immature DC survival in the periphery. Therefore, we Multiple isoforms of the bcl-x gene have been described to date, sought to specifically silence the bcl-xL isoform in skin-resident chief among them being the antiapoptotic bcl-xL and the proapop- DC using siRNA oligonucleotides. DNA vectors based on the pSi- totic bcl-xS. Because the loxP sites engineered into the genome of lencer backbone (Ambion) were engineered to express RNA hair- the bcl-xfl/fl mouse flank all exons of the gene, cre-mediated dele- pin oligonucleotides complementary to 21-nt targets. We directed tion results in the loss of all bcl-x isoforms. Results thus far support the siRNA oligonucleotides against the 189-bp region of bcl-x the hypothesis that sensitivity to apoptosis, stemming from the loss exon 2 that is alternatively spliced from bcl-xS mRNA, and tested of bcl-xL, results in the diminished frequency of marked cells in them for their ability to specifically reduce expression of bcl-xL by FIGURE 6. siRNA-mediated silencing of bcl-xL isoform supports the role of bcl-xL in maintenance of Ag-bearing DC in dLN. RT-PCR analyses of 293 cells cotransfected with a plasmid expressing a bcl-xL-siRNA and either pBcl-xL or pBcl-xS showed specific silencing of bcl-xL expression (a). No change in expression was seen in parallel cotransfection experiments with a GFP-siRNA plasmid (data not shown). Densities were normalized to -actin levels (data not shown) and are representative of two separate experiments. C57BL/6 mice (n 3) were immunized with plasmids dually expressing -gal with or without bcl-xL-siRNA, or control GFP-siRNA, and analyzed for expression of -gal (b) and annexin V (c) by flow cytometry at 2.5, 4.5, and 6.5 days postvaccination. Cells were gated on the early apoptotic 7-aminoactinomycin D (7AAD)lowCD11c population ( SEM). The percentage of annexin V cells in the pCAG-lacZ-immunized group was used as a baseline to calculate the relative frequency of apoptotic cells in the pCAG-lacZ-bcl-xL-siRNA and pCAG-lacZ-GFP siRNA cohorts.
  6. 6. 4430 bcl-xL IS CRITICAL FOR DC SURVIVAL IN VIVO transient cotransfection with bcl-xL and bcl-xS expression con- structs in human 293 cells. One of two selected siRNAs consis- tently 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 simi- larly produced DNA vaccines containing siRNA oligos targeted against GFP (pCAG-lacZ-GFP-siRNA). To confirm that the defect in Ag-bearing DC survival was due to loss of the bcl-xL isoform, we immunized cohorts of C57BL/6 mice with pCAG-lacZ, pCAG-lacZ-bcl-xL-siRNA, or pCAG-lacZ- GFP-siRNA, and analyzed dLN cells for survival of -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-immu- nized group in comparison with the pCAG-lacZ-immunized group (Fig. 6b). In contrast, the pCAG-lacZ-GFP-siRNA-immunized an- imals 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 signif- icant increase in the frequency of -gal-marked cells undergoing FIGURE 7. Low numbers of bcl-xL-deficient DC in dLN are not attrib- apoptosis in the dLN. At 2.5 days postimmunization, there was a utable to a migration defect. To analyze the migration of bcl-xL-deficient 15.2 3.1% increase in the number of CD11c -gal annexin V DC, C57BL/6 mice were gene gun immunized in the ear (n 3) with cells in the pCAG-lacZ-bcl-xL-siRNA-immunized group over pCAG-lacZ, pCAG-lacZ-bcl-xL siRNA, or pCAG-lacZ-GFP siRNA. At baseline levels in the pCAG-lacZ-immunized group (Fig. 6c). This 1.5 and 2.5 days after immunization, we quantified the absolute number of percentage rapidly increased such that, by day 6.5, the bcl-xL-si- CD11c -gal cells in the dLN (a) or remaining at the immunization site lenced CD11c -gal DC population had a 6-fold higher fre- (b). Cells were gated on the early apoptotic 7-aminoactinomycin D quency of apoptotic cells. By contrast, the untransfected (7AAD)lowCD11c population ( SEM). , Significant difference (p CD11c -gal DC population in the pCAG-lacZ-bcl-xL-siRNA- 0.05) from pCAG-lacZ cohort. 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 im- To confirm the migration of the CD11c -gal cells, we ana- munization, and not due to a systemic phenomenon. These results lyzed the draining auricular lymph nodes for the presence of Ag- are in full agreement with the data obtained from cre-mediated bearing DC. Similar to the results observed with immunization of bcl-x excision in DC of bcl-xfl/fl mice, indicating that it was the loss the abdominal skin, we found reduced numbers of bcl-xL-deficient, of the antiapoptotic bcl-xL isoform that produced the survival de- CD11c -gal DC in the dLN at both time points (Fig. 7b). In fect in bcl-x-deficient, migrating CD11c DC. 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 Migration of bcl-xL-deficient DC is not impaired CD11c -gal DC in the dLN mirrored a concomitant decrease in The data presented in Figs. 4 and 6 suggest that the inability to CD11c -gal DC at the immunization site of both control elicit Ag-specific immune responses is due to the heightened sen- groups. The corresponding bcl-xL-deficient population showed a sitivity of bcl-x-deficient DC to apoptosis. However, it is also pos- similar reduction in numbers of Ag-bearing DC at the immuniza- sible that the low numbers of CD11c -gal cells in the dLN may tion site at day 2.5; however, there were persistently low numbers also be due to a defect in migration from the skin. To address this of CD11c -gal cells in the dLN, due to higher levels of apo- issue, we analyzed bcl-xL-deficient DC at the immunization site. ptosis. Taken together, these data indicate that bcl-xL-deficient DC We chose to immunize the ears, because isolation of DC from ear are capable of migration from the periphery, and support the hy- skin as well as from the draining auricular lymph nodes is highly pothesis that impaired longevity of the Ag-bearing DC in the dLN tractable. Briefly, we gene gun immunized cohorts of C57BL/6 causes the failure to elicit immune responses. 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 Delivery of bcl-xL in trans complements bcl-x deficiency the absolute number of CD11c -gal cells that remained at the To further confirm that the bcl-xL isoform is indeed critical for immunization site or that migrated to the dLN. There was no sig- mature DC survival, we attempted to rescue the phenotype ob- nificant difference among the three groups in the absolute numbers served with bcl-x-deficient DC by providing bcl-xL in trans. bcl- of Ag-bearing cells per ear at either time point. Although 1000 xfl/fl mice were immunized with gene gun bullets that had been CD11c -gal cells were still present at the immunization site at cocoated with the pCMV-Cre-CAG-lacZ DNA vector, along with day 1.5, only 250 remained by day 2.5 (Fig. 7a). We also ana- a second plasmid encoding either bcl-xL cDNA, or a mutant bcl-xL lyzed the DC at the immunization site for evidence of apoptosis by (bcl-xL mt7) modified in the BH1 domain to abrogate its antiapop- annexin V staining. We found no difference among the three totic function (29). At 4.5 days after immunization, we evaluated groups in levels of annexin V in the CD11c -gal population CD11c cells from the dLN for their expression of -gal and isolated from the ear at both time points (data not shown), sug- annexin V. Complementation of cre-mediated, bcl-x deficiency gesting that DC were not dying at the immunization site, but had with wild-type bcl-xL, but not bcl-xL mt7, restored the numbers of migrated away from the skin. -gal-marked CD11c DC in the dLN (Fig. 8a). Furthermore, the
  7. 7. The Journal of Immunology 4431 diated 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 antiapop- totic factors such as bcl-2 and bcl-xL in vivo enhances mature DC longevity in secondary lymphoid tissues, and correspondingly in- creases 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 pro- teins (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 antiapop- totic protein bcl-2, and has also been shown to prolong the lon- gevity and Ag-presenting capacity of DC (12, 32). Although it is possible that both bcl-xL and bcl-2 fulfill redundant roles in pro- tection from cell death, gene microarray studies detected a signif- icant increase in bcl-xL expression immediately after DC matura- tion, whereas bcl-2 remained at basal levels (13). Additionally, Kim et al. (29) found that Ag-specific immunostimulation by DNA FIGURE 8. bcl-x deficiency in DC can be rescued by complementation vaccines was most optimally enhanced by engineering constructs with bcl-xL in trans. bcl-xfl/fl mice (n 3) were immunized with gene gun to coexpress bcl-xL, rather than other antiapoptotic proteins such bullets coated with pCAG-lacZ alone or pCMV-Cre-CAG-lacZ alone, or as bcl-2. Indeed, our studies show that bcl-xL-deficient DC are with bullets cocoated with pCMV-Cre-CAG-lacZ and pBcl-xL, or cocoated unable to maintain their survival in the dLN, implying that the loss with pCMV-Cre-CAG-lacZ and pBcl-xL mt7. At 4.5 days after immuni- of this antiapoptotic gene is sufficient to drive these cells to an zation, dLN were isolated and analyzed for expression of -gal (a) and accelerated death. annexin V (b). Numbers represent absolute number of -gal cells ( SEM) or percent change in annexin V cells ( SEM) in the 7-amino- Recent reports (35–37) have suggested that the initiation of T actinomycin D (7AAD)lowCD11c -gal -gated population. 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 addition of wild-type bcl-xL was sufficient to protect the trans- interaction between T cell and DC, the T cell may be able to fected DC from apoptotic death, showing a 21.55 6.8% reduc- autonomously differentiate and exert effector functions. However, tion in the percentage of annexin V cells in the CD11c -gal our studies seem to suggest that survival of Ag-bearing DC is population (Fig. 8b). However, complementation with the mutant critical for the generation of immune responses. bcl-x-deficient DC bcl-xL did not produce any significant change in the level of apo- were still capable of migration from the immunization site, al- ptotic cells. Thus, rescue of bcl-x deficiency in CD11c DC was though they were present in the dLN at much fewer numbers due accomplished by delivery of wild-type bcl-xL in trans. to higher levels of apoptosis. At the peak of migration (2.5 days after gene gun immunization) (3), there were only 1600 Ag- Discussion bearing skin-derived DC per draining superficial inguinal lymph The longevity of Ag-bearing DC in secondary lymphoid tissues is node. Indeed, not only were the frequencies of marked cells in the thought to be a major determinant of the potency of the immune dLN consistently lower, the rate of decline of the bcl-x-deficient response generated. Prolonged survival would increase the chance DC population was much more rapid than that seen in the mice that the DC will make contact with and stimulate the small number immunized with the tracking Ag alone. By day 6.5, the absolute of Ag-specific naive T cells. Previous work has implicated anti- number of CD11c -gal cells had dropped to 250 per lymph apoptotic molecules, such as bcl-xL, in DC survival (11, 12, 30 – node, whereas 5000 could be detected in the control group. The fact 32). Our studies demonstrate the critical importance of bcl-xL in that immune responses were so markedly diminished by the decrease DC longevity and in the magnitude of generated immune re- in the number of -gal-marked, bcl-x-deficient DC in the dLN sponses. The absence of bcl-x in Ag-bearing DC resulted in an suggests that there may be a threshold number of Ag-bearing DC inability to elicit adaptive immunity, as seen in assays of Ag-spe- required to induce acquired immunity. This may be due to the cific T cell proliferation and Ab generation. Biolistic delivery of a inability of the few DC to find the small number of Ag-specific T plasmid dually expressing cre recombinase and -gal into a con- cells. Recent work by our laboratory has shown that previous re- ditional bcl-x knockout mouse (bcl-xfl/fl) can render skin-resident ports enumerating the peak frequencies of Ag-bearing DC had un- DC deficient in bcl-x, and mark them with -gal. Using this strat- derestimated the actual numbers by 100-fold (3), which raises egy, we showed that bcl-x-deficient DC were capable of matura- the possibility that optimal immune responses require a higher tion and migration to dLN. However, their numbers were progres- number of DC than was achieved in the environment of bcl-x sively decreased due to apoptosis, signifying that this gene is deficiency. involved in the survival of mature DC. The lack of immunostimu- The results presented in this study have implications for the lation is thus not due to an intrinsic defect in the DC, but is instead rational design of DC-based vaccine regimens. As vaccine-medi- due to the failure to maintain adequate numbers of Ag-bearing ated immunity is thought to rely upon APC, especially DC, it is APC in the dLN. In addition, the concordance of the effects seen important to find ways by which DC-driven Ag-specific responses with siRNA-mediated bcl-xL silencing with the results of cre-me- can be heightened. Previous studies have attempted to ameliorate
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