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• W2013355912 abstract "Non-homeostatic tissue apoptosis in vivo has been shown to induce inflammatory responses and facilitate the cross-presentation of proteins within apoptotic bodies. We hypothesize that in the presence of foreign antigens, the apoptotic-inflammatory process improves immune priming; further, molecules that trigger apoptosis may be adapted for use as immune adjuvants. One very attractive molecule in this context is the tumor necrosis factor receptor (TNFR) family molecule DR5/TRAIL-receptor 2. We show a significant improvement in CD8+ T-cell mediated vaccine immunity with the use of death receptor-5 (DR5) as an immune adjuvant, a property that is correlated with the activation of caspases-8 (casp8) and dependent on its ability to induce apoptosis in vivo. Non-homeostatic tissue apoptosis in vivo has been shown to induce inflammatory responses and facilitate the cross-presentation of proteins within apoptotic bodies. We hypothesize that in the presence of foreign antigens, the apoptotic-inflammatory process improves immune priming; further, molecules that trigger apoptosis may be adapted for use as immune adjuvants. One very attractive molecule in this context is the tumor necrosis factor receptor (TNFR) family molecule DR5/TRAIL-receptor 2. We show a significant improvement in CD8+ T-cell mediated vaccine immunity with the use of death receptor-5 (DR5) as an immune adjuvant, a property that is correlated with the activation of caspases-8 (casp8) and dependent on its ability to induce apoptosis in vivo. The manner in which apoptotic cells are treated by the adaptive immune system remains controversial. Apoptotic bodies have been shown to transfer proteins/Ag to phagocytes, including to the dendritic cell (DC),1Heath WR Belz GT Behrens GM Smith CM Forehan SP Parish IA et al.Cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens.Immunol Rev. 2004; 199: 9-26Crossref PubMed Scopus (600) Google Scholar but it is unclear whether these Ag are exclusively utilized to maintain tolerance2Sauter B Albert ML Francisco L Larsson M Somersan S Bhardwaj N Consequences of cell death: exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells.J Exp Med. 2000; 191: 423-434Crossref PubMed Scopus (1224) Google Scholar,3Hugues S Mougneau E Ferlin W Jeske D Hofman P Homann D et al.Tolerance to islet antigens and prevention from diabetes induced by limited apoptosis of pancreatic β cells.Immunity. 2002; 16: 169-181Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar or whether they can also be used for immune priming.4Albert ML Sauter B Bhardwaj N Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs.Nature. 1998; 392: 86-89Crossref PubMed Scopus (2039) Google Scholar,5Chattergoon MA Kim JJ Yang JS Robinson TM Lee DJ Dentchev T et al.Targeted antigen delivery to antigen-presenting cells including dendritic cells by engineered Fas-mediated apoptosis.Nat Biotechnol. 2000; 18: 974-979Crossref PubMed Scopus (109) Google Scholar,6Feng H Zeng Y Graner MW Katsanis E Stressed apoptotic tumor cells stimulate dendritic cells and induce specific cytotoxic T cells.Blood. 2002; 100: 4108-4115Crossref PubMed Scopus (135) Google Scholar,7Horwitz MS Ilic A Fine C Rodriguez E Sarvetnick N Presented antigen from damaged pancreatic, β cells activates autoreactive T cells in virus-mediated autoimmune diabetes.J Clin Invest. 2002; 109: 79-87Crossref PubMed Scopus (123) Google Scholar The manner in which apoptotic bodies are formed appears to determine whether they are immunostimulatory or immunosuppressive. Apoptotic bodies formed by cell stressors, extrinsic signaling, or in the presence of inflammatory molecules appear to be stimulatory, whereas apoptotic bodies formed after DNA damage appear to be tolerizing.2Sauter B Albert ML Francisco L Larsson M Somersan S Bhardwaj N Consequences of cell death: exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells.J Exp Med. 2000; 191: 423-434Crossref PubMed Scopus (1224) Google Scholar,8Jenne L Arrighi JF Jonuleit H Saurat JH Hauser C Dendritic cells containing apoptotic melanoma cells prime human CD8+ T cells for efficient tumor cell lysis.Cancer Res. 2000; 60: 4446-4452PubMed Google Scholar,9Basu S Binder RJ Suto R Anderson KM Srivastava PK Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-κ B pathway.Int Immunol. 2000; 12: 1539-1546Crossref PubMed Scopus (1086) Google Scholar,10Fadok VA Bratton DL Rose DM Pearson A Ezekewitz RA Henson PM A receptor for phosphatidylserine-specific clearance of apoptotic cells.Nature. 2000; 405: 85-90Crossref PubMed Scopus (1257) Google Scholar,11Gallucci S Lolkema M Matzinger P Natural adjuvants: endogenous activators of dendritic cells.Nat Med. 1999; 5: 1249-1255Crossref PubMed Scopus (1394) Google Scholar These findings suggest that apoptotic bodies can be utilized to deliver Ag to DC in an immunostimulatory form for the purpose of vaccination. Death receptor-5 (DR5), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 2 or tumor necrosis factor receptor-superfamily (TNFR-SF)-10B, is a 411 amino-acid protein that activates caspases-8 (casp8) mediated apoptosis. The primary 4.4 kb transcript of DR5 is found in many tissues of relevance to the immune system, including the lungs, gut, and secondary lymphoid tissue;12Sheridan JP Marsters SA Pitti RM Gurney A Skubatch M Baldwin D et al.Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors.Science. 1997; 277: 818-821Crossref PubMed Scopus (1533) Google Scholar,13Walczak H Degli-Esposti MA Johnson RS Smolak PJ Waugh JY et al.TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL.EMBO J. 1997; 16: 5386-5397Crossref PubMed Scopus (1020) Google Scholar however, DR5 expression has not been widely documented. Notwithstanding this limitation, the role of DR5 in the immune system is of considerable interest because its only known ligand, TRAIL, is expressed by several tumors14Pan G O'Rourke K Chinnaiyan AM Gentz R Ebner R Ni J et al.The receptor for the cytotoxic ligand TRAIL.Science. 1997; 276: 111-113Crossref PubMed Scopus (1562) Google Scholar,15Smyth MJ Takeda K Hayakawa Y Peschon JJ van den Brink MR Yagita H Nature's TRAIL—on a path to cancer immunotherapy.Immunity. 2003; 18: 1-6Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar raising the possibility that like FasL, TRAIL is used in immune surveillance. Like FasL the expression of TRAIL is highly restricted; only natural killer (NK) subsets, and freshly isolated blood DC have been observed to naturally express TRAIL.16Smyth MJ Cretney E Takeda K Wiltrout RH Sedger LM Kayagaki N et al.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) contributes to interferon γ-dependent natural killer cell protection from tumor metastasis.J Exp Med. 2001; 193: 661-670Crossref PubMed Scopus (432) Google Scholar However, TRAIL expression can be induced on activated CD8+ T cells,17Mirandola P Ponti C Gobbi G Sponzilli I Vaccarezza M Cocco L et al.Activated human NK and CD8+ T cells express both TNF-related apoptosis inducing ligand (TRAIL) and TRAIL receptors, but are resistant to TRAIL-mediated cytotoxicity.Blood. 2004; 104: 2418-2424Crossref PubMed Scopus (147) Google Scholar,18Ochi M Ohdan H Mitsuta H Onoe T Tokita D Hara H et al.Liver NK cells expressing TRAIL are toxic against self hepatocytes in mice.Hepatology. 2004; 39: 1321-1331Crossref PubMed Scopus (117) Google Scholar on peripheral blood T cells exposed to interferon-α (IFN-α), β and γ,19Kayagaki N Yamaguchi N Nakayama M Eto H Okumura K Yagita H Type I interferons (IFNs) regulate tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression on human T cells: a novel mechanism for the antitumor effects of type I IFNs.J Exp Med. 1999; 189: 1451-1460Crossref PubMed Scopus (435) Google Scholar on NK treated with IFN, interleukin-2 and interleukin-15,20Kayagaki N Yamaguchi N Nakayama M Takeda K Akiba H Tsutsui H et al.Expression and function of TNF-related apoptosis-inducing ligand on murine activated NK cells.J Immunol. 1999; 163: 1906-1913PubMed Google Scholar monocytes and DC differentiated ex vivo by IFN.21Griffith TS Wiley SR Kubin MZ Sedger LM Maliszewski CR Fanger NA Monocyte-mediated tumoricidal activity via the tumor necrosis factor-related cytokine, TRAIL.J Exp Med. 1999; 189: 1343-1354Crossref PubMed Scopus (420) Google Scholar,22Fanger NA Maliszewski CR Schooley K Griffith TS Human dendritic cells mediate cellular apoptosis via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).J Exp Med. 1999; 190: 1155-1164Crossref PubMed Scopus (358) Google Scholar This regulation of TRAIL expression by inflammatory mediators suggests there exists a role for DR5 in the immune response. We report here, that (i) DR5/TRAIL-receptor 2 is a potent vaccine adjuvant. Further, (ii) adjuvant activity is dependent on the proapoptotic death domain (DD) ability to induce apoptosis. (iii) We also demonstrate that apoptotic fragments generated after casp8, and not caspases-9, triggering induce DC maturation. Our studies suggest a new role for the TRAIL-DR5 system as a mechanism employed by the innate immune system for surveillance and activation of adaptive immune responses. We studied the ability of three DD containing TNFR-SF members (Fas, TNFR-1 and DR5) to enhance cytotoxic T-lymphocyte (CTL) priming. Mice were immunized with pcEnv alone, or in combination with pDR5, pFas, pTNFR-1 or the vehicle plasmid and human immunodeficiency virus-1 gp160-specific CTL were measured as described in Materials and Methods. In agreement with previous observations pcEnv immunization primes CTLs that kill targets infected with recombinant vaccinia expressing human immunodeficiency virus gp160 (ref. 5Chattergoon MA Kim JJ Yang JS Robinson TM Lee DJ Dentchev T et al.Targeted antigen delivery to antigen-presenting cells including dendritic cells by engineered Fas-mediated apoptosis.Nat Biotechnol. 2000; 18: 974-979Crossref PubMed Scopus (109) Google Scholar) when compared to targets infected with wild-type vaccinia at effector/target (E/T) ratios of 50:1 and 25:1 (Figure 1a). Coimmunization with pcEnv + pFas increases Ag-specific CTL lysis twofold to threefold over mice primed with pcEnv alone, and fivefold over naïve mice (Figure 1b) while pcEnv + pTNFR-1 increased CTL twofold over pcEnv (Figure 1d). The best result was obtained with the pDR5; a threefold to fivefold increase in Ag-specific CTL at E/T 100:1, 50:1, and 25:1 was observed when compared with pcEnv immunized mice and sevenfold compared to naïve mice (Figure 1c). These data indicate that DR5 is the most potent immune adjuvant among DD containing TNFR-SF members. We compared this activity to non-apoptotic, non-DD containing TNFR-SF molecules. Both pRANK (Figure 1e) and pOx40 (Figure 1f) failed to enhance CTL priming. In each case the CTL activity was indistinguishable from that of mice immunized with pcEnv alone. Depletion of CD8+ cells effectively abolished Ag-specific lysis of labeled targets (not shown). The improvement in CTL when priming with pDR5 was correlated with an increase in Ag-specific CD8+ T-cell frequency. Splenocytes from immunized mice were restimulated in vitro with rgp160 (not shown) or a peptide pool of 15-mer peptides spanning the gp160 protein in an enzyme-linked immunosorbent spot assay. The frequency of IFN-γ secreting T cells was quantified as described in Materials and Methods. Among splenocytes restimulated with the peptide pool, three immunizations with pcEnv induced 271 ± 85 IFN-γ spot-forming units (SFU)/106 cells (Figure 1g). The pTNFR-1 and pFas adjuvants increased this frequency 1.5-fold (420 ± 135 SFU/106 cells) and 2.3-fold (636 ± 72 SFU/106 cells), respectively, when compared with pcEnv alone (P < 0.005). However, when coimmunized with pDR5, gp160-specific IFN-γ secreting T cells increased to 1571 ± 85 SFU/106 cells, representing a 5.7-fold increase over the number of T cells primed by immunization with pcEnv. As in the killing assay, no improvement in vaccine response was observed when splenocytes from pcEnv+pRANK or pcEnv + pOx40 groups were restimulated with rgp160 or the gp160-peptide pool (Figure 1h). Recall proliferation to rgp160 was also tested in immunized animals. A comparison of proliferation indices showed an increase in recall proliferation of approximately twofold with pFas [stimulation index (SI) = 6.2 ± 2.5] and fourfold with pDR5 (SI = 11.0 ± 3.9) in comparison to pcEnv alone (SI = 3.1 ± 1.2) (P < 0.05) (Figure 1i). Interestingly pOx40 also improved recall proliferation to rgp160 approximately fourfold (Figure 1j). These studies demonstrate that DR5 has an unusual potency among TNFR-SF members in directing the expansion of CD8+ and CD4+ T-cell responses. To determine whether apoptosis is central to the adjuvant properties of DR5 we constructed a 630 base pair (bp) mutant [(truncated DR5 (tDR5)] in which the cytoplasmic tail was truncated, thereby removing the DD and its ability to interact with procaspases. This truncation should not have affected assembly or trimerization, which is related to the transmembrane and extracellular domains. The expression of both ptDR5 and pDR5 constructs was then tested by in vitro translation (Figure 2a). As shown, pDR5 encodes a 30 kd protein, and ptDR5 encodes an 18 kd mutant. We confirmed that both constructs express to similar levels (Figure 3b); then the biological activity of pDR5 and ptDR5 was investigated by transfecting Rhabdomyosarcoma cells and directly measuring caspase activity and cell viability. Expression of the pDR5 leads to apoptosis of transfected cells with decline in expansion rate of the pDR5 cultures observed as early as at 18 hours (data not shown). At 36 hours, 29.6% of the pVax transfected cells captured a fluorescein isothiocyanate–labeled caspase substrate VAD-FMK (Figure 3a), while 70.4% failed to bind the caspase substrate. In cultures transfected with pDR5 the live/dead was reversed at 36 hours: 60.8% being VAD-FMK+ and 39.2% VAD-FMK- (Figure 3e). ptDR5 transfected cultures were similar to the pVax transfected cultures indicating that the proapoptotic ability of DR5 is lost by the truncation (Figure 3i). This effect was confirmed with the addition of propidium iodide to cultures at 36 hours; 20.7, 49.3, and 20.6% of cells in pVax, pDR5, and ptDR5, respectively were permissive to propidium iodide indicating disruption of their plasma membranes (Figure 3b, f and j).Figure 3Deletion of the death receptor-5 (DR5) cytoplasmic tail leads to a loss of proapoptotic function: The DR5 tail deletion mutant was tested for proapoptotic function in vitro and in vivo. RD cells were transiently transfected with the vector backbone pVax (a, b), the wild-type pDR5 (e, f) and the mutant ptDR5 (i, j). Thirty-six hours after transfection the cells were incubated with the fluorescein isothiocyanate–labeled caspase substrate VAD-FMK for 15 minutes (a, e, i), washed extensively and then analyzed by flow cytometry. Immediately prior to analysis, Propidium iodide (PI) was added to further demonstrate dead cells in the culture (b, d, f). The gates in panels a, e, and i, refer to the percentage of cells retaining a high and low level of the fluorescent caspase substrate. While the gates in panels b, f, and j refer to populations within the culture that are highly permissive or non-permissive to PI. The wild-type and truncated DR5 were also tested for their ability to induce apoptosis, in vivo. Animals were immunized with pcEnv in combination with pVax (c, d), pDR5 (g, h) and ptDR5 (k, l) as previously described. Five-days after immunization the animals were sacrificed and the muscle at the site of immunization was harvested and cryo-sectioned. Apoptotic activity was measured by terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, in situ of the harvested tissue. Panels c, g, and k show tissue sections that were stained with 4', 6-Diamidino-2-phenylindole (DAPI) to reveal the location of nuclei within the muscle fibers. Panels d, h, and l show consecutive tissue sections stained by TUNEL as described in methods.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The loss of function was also confirmed in vivo; muscles isolated from immunization sites on the fifth day postimmunization were probed with 4′,6-diamidino-2-phenylindole and developed by terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), see Materials and Methods. In mice immunized with pcEnv alone no TUNEL staining was observed (Figure 3c and d), suggesting that the immunogen plasmid and immunization process does not lead to acute tissue damage. Addition of pDR5 leads to significant cell death at the immunization site, corresponding to large areas of TUNEL positive cells (Figure 3g and h). This was further confirmed by immunization with pFas, which also induces large areas of TUNEL positive cells at the immunization site (data not shown). As predicted, muscles immunized with ptDR5 were TUNEL negative (Figure 3k and i). Taken together these data suggest that the DD truncation generates a non-signaling, non-apoptotic mutant. To determine whether pDR5 adjuvancy is related to its proapoptotic ability, we compared pDR5 and ptDR5 as adjuvants. Splenocytes from immunized mice were tested for gp160-specific CD8+ T-cell frequency, CTL and CD4+ recall proliferation as described previously. As in previous assays, priming with pDR5 generates strong lytic activity to vaccinia-gp160 infected targets, greater than 60% at E/T 100:1 (Figure 4a). In mice primed with ptDR5, gp160-specific lytic activity was similar to animals immunized with pcEnv alone (<25% at E/T = 100:1). These differences correlated with Ag-specific T-cell frequency; pcEnv+pDR5 increased Ag-specific IFN-γ ELISpot (1381 ± 172 SFU/106 splenocytes) while truncation of the DD returns the gp160-specific IFN-γ ELISpot to the level elicited by pcEnv alone: 475 ± 197 SFU/106 splenocytes vs. 420 ± 135 SFU/106 splenocytes (Figure 4b). Lymphocyte proliferation assay was also reduced from a SI of 14.65 ± 2.61 with pcEnv + pDR5, to SI = 3.91 ± 0.99 with pcEnv + ptDR5 (Figure 4c). This suggests that the adjuvant activity for both CD4+ and CD8+ T cells is related to the DD function. The longevity of the immune response after priming with DR5 was studied in animals immunized with pHA, pHA + pDR5 or pHA + ptDR5 as described. At days 7, 90, and 150 postimmunization, Ag-specific CD8+ T-cell frequency was tested by ELISpot after stimulation with live Influenza. As observed previously with pcEnv, on each postimmunization day the ratio of hemagglutinin (HA)-specific CD8+ T cells in animals primed with pDR5 vs. ptDR5 was consistently more than threefold (Figure 5a). This was correlated with protective immunity using an influenza challenge model. Groups of 20 animals (two cohorts) were vaccinated with pHA, pHA + pDR5 or pHA + ptDR5. The animals were rested until 147 days postimmunization when one cohort was depleted of CD8+ cells as described in Materials and Methods. Depleted and non-depleted cohorts were challenged 3 days later with 1 HA unit live Influenza A PR8/34 (day 150). Non-depleted, naïve animals became infected rapidly, showing rapid weight loss and labored breathing within 3 days postinfection. These animals were followed for 12 days by which time the animals had lost approximately half of their total body weight (Figure 5b). Animals immunized with pHA alone show similar weight loss characteristics initially, but then move along a more protracted weight loss curve eventually reaching a nadir ∼ 7.06 ± 1.89 g representing a 25–30% loss in total body weight. Animals receiving pHA + ptDR5 followed a very similar weight loss curve as those animal receiving pHA alone. While these animals only reach a nadir of 5.38 ± 1.56 g (∼20% loss in total body weight), the behavior of the cohorts is not statistically different from the cohorts immunized with pHA alone at any time-point. Remarkably, the cohort receiving pHA+pDR5 completely controlled the infection. In addition to showing no significant weight loss when compared to their prechallenge weight, there were also no visible signs of infection. Flu specific antibodies were not statistically different amongst the groups (not shown) and the depletion of CD8+ cells completely abolished any protection conferred by vaccination (Figure 5c). These studies support a long-term memory benefit to CD8+ T-cell responses generated by DR5. Finally, we established a relationship between DR5 adjuvant properties and casp8 activation. RD cells were transfected with pLacZ and pDR5, pCasp8, or pCasp9 for 18 hours; they were then lysed and each sample's concentration adjusted to a LacZ activity OD405 nm0.5. The activities of pCasp8 and pCasp9 were easily detected and were associated with the activation of caspase-3 (Figure 6a). Actinomycin-D, which induces active caspase-9 and caspase-3 was used as a control. Notably pDR5 expression led an almost selective activation of casp8 when compared to caspase-9, relative ratio of 18:1; this was associated with caspase-3 activation and apoptosis. We cultured the supernatant fraction from similarly transfected cells with in vitro derived bone marrow DC prepared previously following established protocols.29Inaba K Inaba M Romani N Aya H Deguchi M Ikehara S et al.Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.J Exp Med. 1992; 176: 1693-1702Crossref PubMed Scopus (3331) Google Scholar After 24 hours the DC were analyzed by flow cytometry for the expression of CD86 and major histocompatibility complex-II, both of which are increased during DC maturation. As shown in Figure 6b, apoptotic fragments generated by transfection with pCasp8 stimulate an increase in major histocompatibility complex-II and CD86 in a subset of the population of CD11c+ cells. Fragments generated with pCasp9 do not activate dendritic cells in the same manner despite similar caspase-3 activity. These data suggest that apoptosis triggered by casp8 has different consequences for the immune system as compared to caspases-9. We find that pDR5 which selectively activates casp8, is similarly also associated with DC maturation. TRAIL is a death-inducing molecule expressed by innate immune system cells, including NK and DC16Smyth MJ Cretney E Takeda K Wiltrout RH Sedger LM Kayagaki N et al.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) contributes to interferon γ-dependent natural killer cell protection from tumor metastasis.J Exp Med. 2001; 193: 661-670Crossref PubMed Scopus (432) Google Scholar,20Kayagaki N Yamaguchi N Nakayama M Takeda K Akiba H Tsutsui H et al.Expression and function of TNF-related apoptosis-inducing ligand on murine activated NK cells.J Immunol. 1999; 163: 1906-1913PubMed Google Scholar21Griffith TS Wiley SR Kubin MZ Sedger LM Maliszewski CR Fanger NA Monocyte-mediated tumoricidal activity via the tumor necrosis factor-related cytokine, TRAIL.J Exp Med. 1999; 189: 1343-1354Crossref PubMed Scopus (420) Google Scholar,22Fanger NA Maliszewski CR Schooley K Griffith TS Human dendritic cells mediate cellular apoptosis via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).J Exp Med. 1999; 190: 1155-1164Crossref PubMed Scopus (358) Google Scholar,30Smyth MJ Takeda Hayakawa K Peschon Y JJ van den Brink Yagita H Nature's TRAIL-on a path to cancer immunotherapy.Immunity. 2003; 18: 1-6Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar and activated CD8+ T cells.31Janssen EM Droin NM Lemmens EE Pinkoski MJ Bensinger SJ CD4+ T-cell help controls CD8+ T-cell memory via TRAIL-mediated activation-induced cell death.Nature. 2005; 434: 88-93Crossref PubMed Scopus (510) Google Scholar TRAIL-mediated cytotoxicity is thought to be important in immune surveillance;32Kaliberov S Stackhouse MA Kaliberova L Zhou T Buchsbaum DJ Enhanced apoptosis following treatment with TRA-8 anti-human DR5 monoclonal antibody and overexpression of exogenous Bax in human glioma cells.Gene Ther. 2004; 11: 658-667Crossref PubMed Scopus (18) Google Scholar however, the role of TRAIL-DR5 in immune priming is not well defined. Furthermore, there is little understanding of a link between DR5-apoptosis and adaptive immunity. Although limited, TRAIL expression is well suited to facilitate the delivery of intracellular antigens to DC and the adaptive immune system. We postulate that infected cells might upregulate their expression of DR5, thus becoming targets for killing and transfer of antigens to DC. Alternatively, effectors of the innate immune system such as NK cells, kill infected cells thereby generating apoptotic fragments that can be presented by DC. This would imply that the upregulation of DR5 is carefully controlled and potentially linked to cell stress or other viral sensors. These models suggest that casp8 mediated apoptosis may have different immune consequences as compared to caspases-9 mediated apoptosis (requiring no external stimuli), perhaps leading to the release of immune activating factors that fully capacitate DC (Figure 6). Such a system would allow the immune system to distinguish between homeostatic and non-homeostatic apoptosis and react where appropriate. Perhaps this is in part the reason that viruses have developed multiple mechanisms to prevent apoptosis of infected cells.33Benedict CA Norris PS Ware CF To kill or be killed: viral evasion of apoptosis.Nat Immunol. 2002; 3: 1013-1018Crossref PubMed Scopus (349) Google Scholar Intriguingly, cross-presentation of viral antigens after infection has been shown to activate DC in a Toll-dependent manner.34Schulz O Diebold SS Chen M Naslund TI Nolte MA Alexopoulou L et al.Toll-like receptor 3 promotes cross-priming to virus-infected cells.Nature. 2005; 433: 887-892Crossref PubMed Scopus (747) Google Scholar,35Datta SK Redecke V Prilliman KR Takabayashi K Corr M Tallant T et al.A subset of Toll-like receptor ligands induces cross-presentation by bone marrow-derived dendritic cells.J Immunol. 2003; 170: 4102-4110Crossref PubMed Scopus (258) Google Scholar Our data shows improved T-cell priming to vaccine antigens with the pDR5 adjuvant. We hypothesize that the adjuvant mechanism of DR5 could involve two separate and complementary mechanisms. When plasmids alone are injected, myocytes at the site of immunization are transfected and become the major source of antigen. However, this protein is largely sequestered in myocytes and is only released upon the arrival of primed effectors; these cells view the myocyte as “infected” and kill the cell. Thus, in the absence of adjuvants like DR5 most if not all antigen presentation and immune priming probably occurs by direct transfection of DC with minor contributions from cross-priming pathways.36Porgador A Irvine KR Iwasaki A Barber BH Restifo NP Germain RN Predominant role for directly transfected dendritic cells in antigen presentation to CD8+ T-cells after gene gun immunization.J Exp Med. 1998; 188: 1075-1082Crossref PubMed Scopus (486) Google Scholar,37Chattergoon MA Robinson TM Boyer JD Weiner DB Specific immune induction following DNA-based immunization through, in vivo transfection and activation of macrophages.J Immunol. 1998; 160: 5707-5718PubMed Google Scholar When DR5 is expressed, the transfected myocyte is inclined to undergo apoptosis (Figure 3) by mechanisms that involve the overexpression of DR5 and ligand triggering. This generates Ag-containing apoptotic fragments, which can be internalized by DCs that were not among the cohort that were directly transfected. Thus, with DR5 the contribution of the cross-presentation pathway may be significant to immune priming. We tested this hypothesis by constructing the truncation mutant that was unable to initiate apoptosis (Figure 3). We expected that if apoptosis were central to the adjuvant activity of DR5, the mutant would be functionally inactive and lose all adjuvant properties. Indeed this was found to be the case when ptDR5 was used, since Ag-specific CD8+ T-cell priming was indistinguishable in animals given the plasmid immunogen alone (Figures 3 and 4). Thus, tissue injury and apoptosis may be intimately linked to the recognition of the vaccine antigen. Extending these studies, we have found that several DD containing TNFR-SF members are also DNA vaccine adjuvants; however, none are as potent as DR5. TNFR-1, Fas, DR5 and nerve growth factor research (not shown) induce higher Ag-specific CTL (Figure 1) but DR5 induced robust CTL activity more than fivefold the level induced by the pcEnv plasmid alone and seven times the level in unprimed mice. Further, this effect was consistent in two other Ag systems tested, human immunodeficiency virus-1 gag (data not shown) and Influenza A/PR8 HA (Figure 5a). Again, the adjuvant effect of DR5 was observed when the number of Ag-specific T cells was counted. Interestingly, the non-apoptotic, TRAF interacting molecules Ox40 and RANK as well as CD40 and 4-1BB (not shown) did not increase CTL or responder frequency. It appears that the adjuvant property of TNFR-SF segregates with the DD motif. This result is particularly interesting as it suggests that non-homeostatic apoptosis of Ag bearing cells in vivo serves as a mechanism for the activation of DC and CD8+ T-cell responses and may be an important adju" @default.
• W2013355912 created "2016-06-24" @default.
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• W2013355912 date "2008-02-01" @default.
• W2013355912 modified "2023-09-25" @default.
• W2013355912 title "DR5 Activation of Caspase-8 Induces DC Maturation and Immune Enhancement In Vivo" @default.
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• W2013355912 doi "https://doi.org/10.1038/sj.mt.6300373" @default.
• W2013355912 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18087262" @default.
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