1Center for Cell and Gene Therapy, 2Department of Immunology, 3Department of Molecular and Human Genetics, and 4Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.
Address correspondence to: Si-Yi Chen, Center for Cell and Gene Therapy, Alkek Building N1004, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA. Phone: (713) 798-1236; Fax: (713) 798-1083; E-mail: sychen@bcm.tmc.edu.
J Clin Invest. 2006;116(1):90–100. https://doi.org/10.1172/JCI26169.
The mechanisms utilized by professional APCs to sense microbes and initiate immune responses has been well studied (1–3), largely because of the critical role of APCs such as DCs in initiating and regulating immune responses and their therapeutic potential (4–6). When DCs encounter proinflammatory stimuli, such as microbial products, the maturation process is initiated and results in proinflammatory cytokine secretion and upregulation of MHC molecules and costimulatory molecules (4, 5). Following maturation and homing to LNs, DCs establish contact with T cells by forming an immunological synapse, where the TCR/MHC interaction and costimulatory molecules congregate in a central area surrounded by adhesion molecules (7). Once activated, CD8+ CTLs can proliferate for several generations and acquire lytic function (8, 9). It has therefore been proposed that the level and duration of peptide-MHC complexes (signal 1) and costimulatory molecules (signal 2) provided by DCs determines the magnitude and fate of an antigen-specific T cell response (10, 11). In addition, cytokines have been implied as a third signal for the activation of T cells by DCs (12, 13).
Little is known about the negative regulatory mechanisms in DCs used to control the magnitude and overactivation of T cell responses, in contrast to the extensive studies on DC activation. Several studies demonstrate that SOCS1 is a negative regulator of LPS-induced macrophage activation and plays an essential role in suppressing systemic autoimmunity mediated by DCs (14–16). SOCS1 is an inducible negative feedback inhibitor of the Jak/Stat signal pathway (17). Homozygous genetic KO of SOCS1 in mice results in neonatal lethality due to uncontrolled IFN-γ signaling (18, 19). SOCS1-KO DCs are hyperactivated and induce aberrant expansion of B and T cells. Recently we found that SOCS1-silenced DCs were able to induce an enhanced antigen-specific CTL response and antitumor activity (20). We have identified SOCS1 as an important negative regulator of antigen presentation by DCs and demonstrated that silencing of SOCS1 enhances antigen presentation by DCs and antigen-specific antitumor immunity (20). In agreement, Hanada et al. reported that immunization with SOCS1-KO DCs induced a hyper–Th1-type immune response and antitumor activities (21).
Immunologic peripheral tolerance to self antigen reflects the inability of autoreactive T cells that escape censoring in the thymus to cause autoimmune pathologies. The essential requirement for an effective tumor vaccine is its ability to break self tolerance and induce pathological autoimmune responses against tumors and normal nonessential tissues that express self tumor-associated antigens (self TAAs) (22). DC vaccines have been viewed as 1 of the most promising strategies for tumor vaccination (4, 6, 23, 24). A puzzling paradox is that mature DC immunization can effectively break self tolerance at the cellular level, i.e., activate self antigen–specific CTLs, but rarely causes autoimmune pathologies against normal tissues and tumors (25), suggesting that self tolerance at the host level is still maintained in a host's natural immunosuppressive environment, sustained by various mechanisms such as regulatory T cells and exaggerated by the tumor-derived factors (26, 27), impeding the efficacy and usefulness of DC-based tumor vaccination.
The aims of this study were to investigate the underlying regulatory mechanisms in DCs used to control the magnitude and overactivation of autoreactive CTL responses and to define the requirements for DCs to induce TAA-specific, pathological autoimmune antitumor responses. We investigate autoimmune CTL responses against a natural self antigen, tyrosinase-related protein 2 (TRP2), in syngeneic, WT mice (28). This self TRP2 mouse model may limit the problems associated with the use of transgenic mouse models and/or the use adoptive transfer of high-avidity TCR-transgenic T cells (29). The results of this study demonstrate that SOCS1 functions as an antigen presentation attenuator for the control of self tolerance at the host level through the restriction of IL-12 and the downstream cytokine signaling cascade by SOCS1. This study further establishes a new principle for breaking self tolerance at the host level and inducing self TAA-specific, pathologic antitumor immunity by DC vaccination.
DC immunization and persistent in vivo stimulation with TLR agonists fail to break self tolerance at the host level. We first tested whether DC immunization, plus persistent in vivo TLR signaling, was able to break self tolerance and induce self antigen–specific autoimmune pathologies. We selected this combined immunization protocol because DCs are the most potent professional APCs, and TLR signaling is the most potent means to activate DCs and immune responses (1, 2, 4). We used the mouse melanocyte differentiation antigen TRP2 as a model self antigen because it is naturally expressed in normal melanocytes as well as weakly immunogenic B16 melanoma, and multiple MHC class I epitopes of varying affinity have been identified within TRP2 (28). WT C57BL/6 mice were administered TRP2-pulsed, LPS-matured DCs derived from mouse BM cells. The mice were then stimulated in vivo (i.p.) with TLR agonists to TLR2, -3, -4, and -7 (LPS [TLR4], poly[I:C] [TLR3], peptidoglycan [TLR2], or imiquimod [TLR7]), which are expressed on mouse myeloid DCs (30), for 7 consecutive days. TRP2-specific CTL responses were efficiently induced in mice immunized with WT DCs and TLR agonists as shown by IFN-γ enzyme-linked immunospot (ELISPOT) analysis (Figure 1A). However, despite persistent in vivo TLR stimulation after DC transfer, the development of autoimmune vitiligo, a self-reactive immune response targeted against TRP2 in host melanocytes that is characterized by the development of coat lightening, depigmentation, and/or hair loss, was absent from all immunized mice. Immunization with TRP2-pulsed, matured DCs, followed by in vivo stimulation with various TLR agonists, failed to effectively control tumor progression (Figure 1, B and C). These results indicate that this immunization protocol of mature DC transfer plus persistent in vivo TLR signaling can effectively break self tolerance at the cellular level, i.e. activate self antigen–specific CTLs, but not at the host level, as manifested by the lack of autoimmune pathologies against self antigen–expressing normal tissues and tumor.
Persistent TLR stimulation of DC immunization failed to induce pathological autoimmune response and antitumor immunity. (A) TRP2-specific CTL responses induced by persistent in vivo TLR stimulation and DC immunization. C57BL/6 mice were immunized with TRP2-pulsed (100 μg/ml) and LPS-matured (100 ng/ml) DCs and then stimulated with various TLR agonists daily for 7 consecutive days. Splenocytes pooled from immunized mice (2–3 mice) were subjected to IFN-γ ELISPOT assays. An irrelevant peptide (OVA-I peptide) was used as a negative control. (B and C) Inability to control preestablished B16 tumors by persistent in vivo TLR stimulation and DC immunization. Groups of mice were inoculated s.c. with B16 tumor cells (2.5 × 105) and 3 days later were immunized via the rear foot pad with 1.5 × 106 TRP2 peptide–pulsed (100 μg/ml) DCs with ex vivo LPS maturation. After DC transfer, in vivo TLR agonists were administered i.p. daily for 7 days. Tumor growth and percent survival (n = 5–7 mice/group) curves represent 1 of 3 independent experiments. When their tumor volume reached approximately 2,000 mm3 in size, mice were euthanized and recorded as dead.
Silencing of a signaling inhibitor allows DCs to break self tolerance and induce autoimmune pathologies. To investigate possible molecular restrictions in DCs to prevent autoimmune pathologies, we tested the effect of silencing SOCS1 in DCs on autoimmune responses based upon our previous findings (20). A lentiviral vector, LV-SOCS1-siRNA (eYFP), with the ability to specifically downregulate approximately 90% of SOCS1 mRNA in BM-derived DCs and a control vector, LV-GFP-siRNA (eYFP), were generated as described previously (20). In agreement, the SOCS1 protein expression in the transduced LV-SOCS1-siRNA DCs was significantly reduced, as demonstrated by Western blotting (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI26169DS1). TRP2-pulsed, transduced DCs were transferred into WT C57BL/6 mice. The mice were then stimulated in vivo with or without a low dose of LPS, and CTL responses were measured. As the number of in vivo LPS stimulations was increased, the amount of TRP2-specific CD8+ T cells also increased in SOCS1-siRNA DC mice (Figure 2A). In contrast, the amount of TRP2-specific CD8+ T cells was only marginally increased in GFP-siRNA DC mice, regardless of the number of LPS stimulations, and was consistently lower than that in SOCS1-siRNA DC mice (Figure 2A, left panels). IFN-γ ELISPOT and CTL assays showed similar results (data not shown). The development of autoimmune vitiligo was apparent at 2–3 months after immunization in many of the SOCS1-siRNA DC mice injected with LPS (Figure 2B). In contrast, autoimmune vitiligo was not observed in any of GFP-siRNA DC mice, even with repeated in vivo LPS administrations. Although the mechanisms for the inability of GFP-siRNA DCs to induce stronger antitumor immunity in response to repeated LPS stimulation are still not clear, SOCS1-restricted JAK/STAT signaling, endotoxin tolerance, and/or other mechanisms may contribute to this outcome. Interestingly, we observed that the duration of the presence of SOCS1-siRNA DCs in draining LNs of immunized mice was increased, compared with that of GFP-siRNA DCs (Figure 2C), suggesting that the enhanced response of SOCS1-siRNA DCs to repeated LPS stimulation is, in part, due to the prolonged duration of these DCs in draining LNs. Taken together, these results suggest a critical role of SOCS1 in DCs for maintaining tolerance to self antigens at the host level and a necessity of silencing the signaling inhibitor to induce pathological autoimmune responses by DC immunization.
SOCS1-silenced DCs induced pathological autoimmune responses. (A) TRP2-specific CTL responses induced by SOCS1-silenced DCs. Mice were immunized with TRP2-pulsed (50 μg/ml), LV-transduced WT, or IL-12p35–KO DCs with LPS-induced maturation (100 ng/ml) ex vivo. The mice were then stimulated 0, 1 (day 1), or 3 times (days 1, 4, and 7) i.p. with LPS (30 μg/mouse/injection). Percentages of TRP2-tetramer-PE–positive T cells in the gated CD8+ T cells of splenocytes in mice 2 weeks after immunization are shown from 1 of 3 independent experiments. P < 0.01, GFP-siRNA DCs versus SOCS1-siRNA DCs. (B) Representative autoimmune vitiligo of mice 3 months after immunization with TRP2-pulsed LV-SOCS1-siRNA DCs, followed by LPS stimulation once or 3 times. (C) Duration of DCs in draining LNs. Groups of mice were immunized with LV-transduced DCs, and CD11c+ DCs isolated from the draining LNs (dLN) of the mice at different times were used for RT-PCR analysis of the transgene eYFP marker mRNA. GAPDH was used as internal control. Experiments were repeated twice with similar results. (D and E) Inhibition of preestablished B16 tumors by SOCS1-siRNA DCs. WT, CD4-KO, or CD8-KO C57BL/6 mice were inoculated s.c. with B16 tumor cells (2.5 × 105) and 3 days later were immunized via the rear foot pad with 1.5 × 106 TRP2 peptide–pulsed (50 μg/ml), LV-transduced DCs with ex vivo LPS maturation (100 ng/ml). One day after DC transfer, in vivo LPS was administered i.p. (30 μg/mouse) 1 time. Tumor growth (n = 6 mice/group) and percent survival curves represent 1 of 3 independent experiments. P < 0.01, GFP-siRNA DCs compared with SOCS1-siRNA DCs.
SOCS1-silenced DCs induce effective antitumor immunity capable of controlling preestablished B16 tumors when costimulated with a TLR agonist. We (20) and Hanada et al. (21) have independently shown that SOCS1-siRNA DC or SOCS1-KO DC vaccination of weakly immunogenic (TRP2+) B16 tumor–bearing mice results in a reduction in tumor growth and an increase in survival; however, tumor growth is not fully inhibited, and mice eventually succumb to tumor burden. Therefore, we tested whether SOCS1-silenced DCs more potently induce autoreactive CTL responses capable of controlling the growth of preestablished B16 tumors when boosted in vivo with a TLR agonist such as LPS, a potent inducer of proinflammatory cytokines (1–3). The addition of in vivo stimulation with LPS significantly enhanced the ability to inhibit B16 tumors and promote mouse survival by immunization with SOCS1-siRNA but not GFP-siRNA DCs (Figures 2, D and E). The enhanced antitumor activity was correlated with potent TRP2-specific CTL activities observed in SOCS1-siRNA DC mice (data not shown). By transferring SOCS1-siRNA DCs into CD4- and CD8-KO mice, it was further demonstrated that the antitumor response required both CD8+ and CD4+ cells, although a weak antitumor response was observed in CD4-KO mice (Figures 2, D and E). No apparent toxicity, other than autoimmune vitiligo, was observed in the TRP2-pulsed SOCS1-siRNA DC mice coinjected with LPS more than 6 months after immunization. Histological analysis of major organs and tissues of the immunized mice revealed no pathologic inflammation (data not shown). Collectively, these results suggest that SOCS1-restricted signaling of TLR agonist–induced cytokines in DCs may control their ability to break tolerance at the host level and induce effective antitumor immunity.
Role of SOCS1-restricted cytokine (IL-12) production by DCs in breaking self tolerance at the host level. We next investigated possible mechanisms used by SOCS1 in DCs to regulate antigen presentation by examining its influence on 3 major signals provided to T cells: antigenic peptide/MHC presentation (signal 1), costimulatory molecule expression (signal 2), and/or cytokine production (signal 3). By flow cytometric assays, we consistently found that there were only slightly altered surface levels of costimulatory/inhibitory molecules (B7.1, B7.2, OX40L, CD40, or PDL1) on SOCS1-siRNA DCs compared with those on GFP-siRNA DCs both before and after LPS-induced maturation (data not shown), in agreement with our previous observation (20). Comparable levels of MHC class I and II molecules (signal 1) were also detected on SOCS1-siRNA DCs and GFP-siRNA DCs (data not shown). We further investigated whether peptide immunogenicity (TCR affinity) can influence SOCS1-restricted CTL responses in vivo. Since a high-affinity form (TRP2b) and a low-affinity form (TRP2a) of the TRP2 CTL peptide were identified previously (28), we used these 2 TRP2 peptides to test whether the strength of signal 1 can influence the ability of transduced DCs to control B16 tumor growth. Figure 3A shows that GFP-siRNA DCs loaded with either the low- or high-affinity peptide were unable to induce B16 tumor regression with in vivo LPS stimulation, although GFP-siRNA DCs loaded with the TRP2b peptide showed a marginal effect on tumor growth. In contrast, both SOCS1-siRNA DC groups loaded with the low- or high-affinity TRP2 peptide effectively blocked tumor growth. We investigated TRP2-specific CTL activities in vaccinated mice with IFN-γ ELISPOT. Figure 3B shows that GFP-siRNA DCs loaded with the high-affinity peptide induced stronger IFN-γ responses than did GFP-siRNA DCs loaded with the low-affinity peptide. However, both SOCS1-siRNA DCs loaded with low- or high-affinity peptide induced much stronger IFN-γ responses than GFP-siRNA DCs (P < 0.01). These results show that SOCS1 in DCs does not have a significant impact on the expression of costimulatory/inhibitory molecules or MHC class I and II molecules on DCs; and SOCS1-restricted signaling in DCs plays a more dominant role than self peptide affinity in inducing CTL responses and antitumor immunity.
Effects of SOCS1 silencing on antigen presentation by DCs. (A) Inhibition of preestablished B16 tumor by SOCS1-siRNA DC immunization with TRP2 peptides of different affinities. WT C57BL/6 mice were inoculated s.c. with B16 tumor cells (2.5 × 105) and 3 days later were immunized with 1.5 × 106 TRP2 peptide–pulsed (50 μg/ml; TRP2a or TRP2b), transduced DCs with ex vivo LPS maturation (100 ng/ml). One day after DC transfer, in vivo LPS was administered i.p. (30 μg/mouse) 1 time. Tumor growth curves (n = 6 mice/group) represent 1 of 3 independent experiments. P < 0.01, GFP-siRNA DC compared with SOCS1-siRNA DCs. (B). CD8+ T cell responses induced by SOCS1-siRNA DCs with different TRP2 peptides. CD8+ T cells isolated from the pooled splenocytes of immunized mice (2–3 mice) were subjected to IFN-γ ELISPOT assays stimulated with TRP2a or TRP2b peptide (10 μg/ml). An irrelevant peptide derived from ovalbumin was used as a negative control. *P < 0.01 versus SOCS1-siRNA DC plus TRP2a; **P < 0.01 versus SOCS1-siRNA DC plus TRP2a.
The above observations and known SOCS1 functions as an inhibitor of Jak/Stat signaling (17) imply a critical role of SOCS1-restricted cytokine signaling and production in controlling T cell responses. We initially tested the importance of several candidate cytokines known to influence CTL activation by using DCs derived from the BM of different KO mice. LV-transduced DCs derived from IL-12p35–KO mice (p35–/– SOCS1-siRNA DCs) were no longer able to inhibit the growth of preestablished B16 tumors (Figures 4, A and B). IFN-γ ELISPOT and CTL assays showed that p35–/– SOCS1-siRNA DCs had a substantially reduced ability to induce TRP2-specific CTL responses compared with WT SOCS1-siRNA DCs (Figure 4C and Supplemental Figure 2). In addition, in vivo stimulation with LPS failed to boost CTL responses induced by p35–/– SOCS1-siRNA DCs as measured by TRP2-tetramer analysis (Figure 2A), and autoimmune vitiligo did not develop in these mice (data not shown). In contrast to the essential role of IL-12 produced by antigen-presenting DCs, antitumor CTL responses were still effectively induced in IL-12p35–KO mice immunized with WT SOCS1-siRNA DCs, suggesting that IL-12 produced by host cells is not required for the induction of CTL responses (Figure 4, A–C, and Supplemental Figure 2). These results suggest that IL-12 production by SOCS1-silenced, antigen-presenting DCs is required for inducing a potent TRP2-specific CTL response and breaking self tolerance at the host level.
The role of IL-12 produced by SOCS1-silenced DCs in breaking self tolerance and inducing antitumor immunity. (A and B) Inability to inhibit preestablished B16 tumor by IL-12–deficient SOCS1-siRNA DCs. WT or IL-12p35–KO mice were inoculated s.c. with B16 tumor cells (2.5 × 105) and 3 days later were immunized with 1.5 × 106 TRP2-pulsed, transduced WT, or IL-12p35–KO DCs with ex vivo LPS maturation. Tumor growth (A) and percent survival (B) (n = 6 mice/group) curves represent 1 of 2 independent experiments. P < 0.01, GFP-siRNA DCs compared with SOCS1-siRNA DCs. (C) CD8+ T cell responses induced by IL-12–deficient SOCS1-siRNA DCs. CD8+ T cells isolated from the pooled splenocytes of immunized WT or IL-12p35–KO mice were subjected to IFN-γ ELISPOT assays. An irrelevant peptide (OVA-I peptide) was used as a negative control. *P < 0.01 versus SOCS1-siRNA DCs.
Prolonged and enhanced production of IL-12 by SOCS1-silenced DCs versus transient and low production by WT DCs. DCs produce significant amounts of heterodimeric IL-12 in response to microbial products and/or CD40 ligand. However, the duration of IL-12 production by stimulated DCs is tightly restricted to a short time period (8–16 hours) (31). We determined the effect of SOCS1 on the concentration and duration of IL-12 produced by DCs following in vitro stimulation. To test this we stimulated SOCS1-silenced DCs or control DCs with LPS alone or in combination with stimulatory anti-CD40 antibody and then measured the production of IL-12. We observed a small but significant (P < 0.05) increase in the production of IL-12p40 by SOCS1-silenced DCs derived from BM culture with GM-CSF and IL-4, compared with control DCs (Figure 5A), which is consistent with a previous observation (32). Strikingly, the expression of IL-12p70 by SOCS1-siRNA DCs was substantially increased after stimulation compared with that by control DCs (Figure 5B), which is consistent with 2 recent reports that SOCS1-KO macrophages produced excessive amounts of IL-12 and other cytokines in response to stimuli and that enhanced levels of IL-12 and other cytokines were found in sera of conditional SOCS1-KO mice (33, 34). Interestingly, BM DCs after culture with only GM-CSF produced less IL-12 (Figure 5A), likely due to the previously described stimulatory effects of IL-4 on IL-12 production by DCs (35). The marked difference in IL-12p70 compared with IL-12p40 secretion is likely due to the upregulation of IL-12p35 expression, which has previously been shown to act as the rate-limiting step for IL-12 heterodimer secretion (36). Indeed, higher levels of IL-12p35 mRNA were also detected in SOCS1-siRNA DCs, compared with control GFP-siRNA DCs (Supplemental Figure 3). We also observed that the levels of the IFN-inducible factors ICSBP and IRF-1 mRNA (37) appeared enhanced in SOCS1-siRNA DCs, while the transcription factors C/EBP-β and c-Rel remained largely unchanged compared with control DCs (Supplemental Figure 3). Consistent with the in vitro data, the expression levels of IL-12p35 as well as other proinflammatory cytokines, IFN-γ and IL-2, were elevated in DCs isolated from the draining LNs of mice receiving SOCS1-siRNA DCs and in vivo LPS stimulation (Supplemental Figure 4). Finally, we found that SOCS1-silenced DCs were able to continuously produce high levels of IL-12p70 for at least 72 hours when stimulated with LPS/anti-CD40 mAb, while control GFP-siRNA DCs produced only low levels of IL-12p70 over the same culture period (Figure 5C). These results indicate that SOCS1 silencing allows DCs to continuously produce increased levels of IL-12p70 in response to inflammatory stimulation.
SOCS1 restricts the level and duration of IL-12 produced by DCs. (A and B) Levels of IL-12p40 (A) and IL-12p70 (B) secreted by DCs (5 × 105 cells/ml) in response to continuous stimulation with LPS (100 ng/ml) with or without plate-coated anti-CD40 mAb (1:1,000 dilution) for 24 hours from 1 of 3 independent experiments. All BM DCs were cultured with mGM-CSF (20 ng/ml) in the presence or absence of mIL-4 (20 ng/ml). *P < 0.01 versus SOCS1-siRNA DCs. (C). Kinetics of IL-12p70 production by DCs (5 × 105 cells/ml) in response to continuous stimulation with LPS (100 ng/ml) and anti-CD40 mAb for 0–72 hours from 1 of 3 independent experiments. All cells were cultured in the presence of both GM-CSF and IL-4. *P < 0.01 GFP-siRNA DCs versus SOCS1-siRNA DCs.
Increased and continuous production of IL-12 by WT DCs is not sufficient to break self tolerance at the host level. To investigate whether overproduction of IL-12 alone may account for the ability of SOCS1-silenced DCs to break self tolerance at the host level, we compared the ability of WT DCs transfected with recombinant adenovirus constitutively expressing biologically active mouse IL-12 cytokine (Ad–IL-12) and SOCS1-silenced DCs to induce antitumor CTL responses. DCs transfected with Ad–IL-12 at an MOI of 300 produced a high level of IL-12p70, comparable to the IL-12p70 level produced by SOCS1-siRNA DCs after stimulation with LPS. Unexpectedly, immunization with mature TRP2-pulsed DCs transfected with different MOIs of Ad–IL-12 failed to control the growth of preestablished B16 tumors (Figure 6A). In agreement, in mice immunized with Ad–IL-12 DCs (300 or 1,000 MOI), only modest increases in CTL responses were observed (Figure 6, B and C). Furthermore, we found that in vivo stimulation with a low dose of IL-12 did not significantly enhance CTL responses and antitumor activities in GFP-siRNA DC mice but significantly enhanced the antitumor CTL responses in SOCS1-siRNA DC mice (Figure 6, A–C), suggesting the enhanced sensitivity of SOCS1-silenced DCs to IL-12 stimulation. Importantly, vitiligo was not induced in mice immunized with Ad–IL-12 DCs or GFP-siRNA DCs with IL-12 coinjection, while it was observed in about 40% of TRP2-pulsed, SOCS1-siRNA DC mice coinjected with a low dose of IL-12 (Supplemental Figure 5). These results indicate that continuous overexpression of IL-12 by WT DCs is not sufficient to break self tolerance at the host level and that SOCS1-restricted signaling of IL-12 in antigen-presenting DCs and the concentration of IL-12 produced by DCs play a critical role in controlling CTL responses and tolerance.
WT DCs constitutively expressing IL-12 were insufficient to overcome self tolerance at the host level. (A) Inability to inhibit preestablished B16 tumors by Ad–IL-12 DCs. BM DCs were transduced with Ad–IL-12 (MOIs of 300 or 1,000) or LV-SOCS-siRNA or LV-GFP-siRNA (MOI of 5). WT mice were inoculated s.c. with B16 tumor cells (2.5 × 105) and 3 days later were immunized with 1.5 × 106 TRP2-pulsed DCs with ex vivo TNF-α maturation (50 ng/ml). After transfer of DCs, IL-12 (1 μg/mouse) was administered i.p. 3 times on days 1, 3, and 5. Tumor growth curves (n = 6 mice/group) represent 1 of 3 independent experiments. (B and C). Antigen-specific CD8+ T cell responses. Splenocytes or CD8+ T cells isolated from the pooled splenocytes of immunized mice were subjected to IFN-γ ELISPOT assays (B) or intracellular IFN-γ staining (C). *P < 0.01 versus SOCS1-siRNA DCs plus IL-12.
Unbridled IL-12 signaling in DCs is required to break self tolerance at the host level. To test the role of IL-12 signaling in DCs for breaking self tolerance at the host level, we compared the CTL responses induced by WT DCs and DCs derived from IL-12 receptor–KO mice (IL-12Rβ2–/–). SOCS1-silenced DCs derived from IL-12Rβ2–/– SOCS-siRNA DCs exhibited a substantially reduced ability to induce TRP2-specific CTLs compared with WT SOCS1-siRNA DCs (Figures 6C and 7B). Furthermore, p35–/– SOCS1-siRNA DCs and IL-12Rβ2–/– SOCS1-siRNA DCs were unable to induce either tumor regression (Figure 7A) or autoimmune vitiligo (data not shown). We found that p35–/– SOCS1-siRNA DCs and IL-12Rβ2–/– SOCS1-siRNA DCs had a substantially reduced ability to induce TRP2-specific CTLs compared with WT SOCS1-siRNA DCs (Figure 7B). To further investigate the role of IL-12 signaling in DCs, we examined the ability of in vivo IL-12 stimulation to enhance CTLs in mice immunized with IL-12Rβ2–/– SOCS1-siRNA DCs. Figures 7, C and D, show that in vivo IL-12 stimulation of IL-12Rβ2–/– SOCS1-siRNA DC mice failed to enhance TRP-specific CTL responses. Collectively, these results indicate that SOCS1-restricted IL-12 signaling in antigen-presenting DCs plays a critical role in controlling antigen presentation and the unbridled signal transduction of IL-12 in antigen-presenting DCs is required to break self tolerance and induce pathological autoimmune responses.
Persistent cytokine signaling in DCs is required to induce pathological autoimmune pathologies. (A) Inability to inhibit preestablished B16 tumors by IL-12p35–/–, IL-12Rβ2–/–, or IFN-γ–/– DCs. BM DCs derived from either WT, IL-12p35–/–, IFN-γ–/–, or IL-12Rβ2–/– mice were transduced with LV-SOCS1-siRNA or LV-GFP-siRNA. WT mice were inoculated s.c. with B16 tumor cells (2.5 × 105) and 3 days later were immunized with 1.5 × 106 TRP2-pulsed, matured DCs, followed by poly(I:C) stimulation i.p. on days 1, 3, and 5. Tumor growth curves (n = 6 mice/group) represent 1 of 3 independent experiments. (B) Reduced potency to induce CTL responses by SOCS1-silenced, IL-12Rβ2–/– DCs or IFN-γ–/– DCs. CD8+ T cells isolated from the pooled splenocytes of mice immunized with DCs derived from WT, IFN-γ–/–, IL-12p35–/–, or IL-12Rβ2–/– mice, followed by poly(I:C) stimulation 3 times, were subjected to IFN-γ ELISPOT assays. *P < 0.01 versus SOCS1-siRNA DCs. (C and D) In vivo IL-12 stimulation failed to enhance the potency of SOCS1-silenced, IL-12Rβ2–/– DCs. Mice were immunized once with WT or IL-12Rβ2–/– DCs (1.5 × 106/mouse) pulsed with TRP2 and matured with TNF-α, followed by IL-12 (i.p.) 3 times. Two weeks later, splenocytes or CD8+ T cells isolated from the pooled splenocytes were subjected to IFN-γ ELISPOT (C) and CTL assays against B16 tumor cells (D). *P < 0.01 versus SOCS1-siRNA DCs.
SOCS1 restricts IL-12 and cytokine signaling cascade in DCs. Our data suggest that silencing SOCS1 may allow the establishment of an unbridled signaling cascade of IL-12 and IL-12–induced downstream cytokines in DCs. To further determine the ability of IL-12 to continuously signal in SOCS1-siRNA DCs, we examined the duration of IL-12 signaling in WT and SOCS1-silenced DCs by analyzing the phosphorylation kinetics of Stat4 (phosphorylated Stat4 [pStat4]), which is primarily used by members of the IL-12 cytokine family (36). Figure 8A shows that Stat4 was persistently phosphorylated for at least 72 hours in SOCS1-silenced DCs after LPS/anti-CD40 stimulation, whereas pStat4 was already at basal levels at 24 hours in GFP-siRNA DCs, in agreement with the continuous secretion of IL-12p70 by SOCS1-silenced DCs in response to LPS/anti-CD40 mAb stimulation shown in Figure 5C. Furthermore, Ad–IL-12–transfected DCs were found to only have low levels of pStat4 at the indicated time points, suggesting that SOCS1 inhibits Stat4 activation in Ad–IL-12 DCs that continuously produced and exposed to IL-12 (Supplemental Figure 6). We then examined whether IL-12 induces the enhanced production of downstream cytokines by SOCS1-silenced DCs. Figure 8B shows that IL-12 stimulation induced substantially higher levels of inflammatory cytokines such as IFN-γ by mature SOCS1-siRNA DCs than by mature GFP-siRNA DCs, suggesting that SOCS1-silenced DCs have an increased sensitivity to IL-12 stimulation. Figure 8C shows that GFP-siRNA DCs and mock-transduced DCs only transiently produced IL-12p70. In contrast, SOCS1-siRNA DCs persistently produced IL-12p70 after stimulation, at levels comparable to Ad–IL-12 DCs (MOI, 300). Importantly, this increased level of IL-12p70 secretion by SOCS1-siRNA DCs was dependent on the expression of the IL-12 receptor (Figure 8C), indicating that the marked increase in IL-12p70 secretion by SOCS1-silenced DCs is likely due to autocrine signaling by IL-12p70. IL-12 stimulation induces IFN-γ production by DCs (38), and IFN-γ was recently found to be hyper-produced by SOCS1-KO DCs in response to LPS (21). Therefore, we examined whether an unbridled cytokine network, not just IL-12, in SOCS1-silenced DCs is collectively responsible for inducing effective anti-tumor immunity. Using IFN-γ–KO DCs, we found that IFN-γ–KO SOCS1-siRNA DCs only induced weak CTL responses and did not control the growth of preestablished B16 tumors (Figure 7, A and B), suggesting an important role of IFN-γ in inducing antitumor CTL responses by SOCS1-siRNA DCs as well. Collectively, these data suggested that SOCS1 restricts cytokine signaling and only allows DCs to activate CTLs in a pulsing or transient fashion, thus limiting autoimmune pathologies. These data further suggest that SOCS1 silencing leads to the unbridled signaling of IL-12 and the downstream cytokine network in antigen-presenting DCs, resulting in the breaking of self tolerance at the host level and pathological autoimmune responses.
Enhanced signaling and sensitivity of SOCS1-silenced DCs to autocrine/paracrine IL-12 stimulation. (A) Western blotting of Stat4 and phosphorylated Stat4 (pStat4) in LV-transduced BM-DCs (1 × 106 cells/ml) after stimulation with LPS and anti-CD40 mAb for 0, 24, 48, or 72 hours. BM DCs were cultured in the presence of GM-CSF and IL-4. (B) IFN-γ levels secreted by LV-transduced, TNF-α–matured WT DCs (1 × 106 cells/ml) 6, 24, or 48 hours after stimulation with recombinant IL-12 (20 ng/ml) from 1 of 3 independent experiments. *P < 0.01 versus SOCS1-siRNA DCs. (C) Levels of IL-12p70 secreted by transduced DCs (1 × 106 cells/ml) derived from WT or IL-12Rβ2–/– mice at different times after stimulation with TLR agonist (LPS, 100 ng/ml) from 1 of 3 independent experiments. All cells were cultured in the presence of GM-CSF and IL-4. *P < 0.01 versus WT SOCS1-siRNA DCs.
This study uncovers a novel regulatory mechanism for the control of pathological autoimmune responses by DCs through the restriction of cytokine signaling and the responsiveness to stimuli by SOCS1. We find that optimal immunization with matured WT DCs loaded with high-affinity self peptide (TRP2) plus persistent TLR signaling effectively activate self-reactive T cells but fail to break self tolerance at the host level. In contrast, SOCS1-silenced DCs effectively break tolerance at the host level and cause self antigen–specific, autoimmune pathologies against normal tissues and tumors. These results indicate that the requirement for breaking self tolerance at the cellular level (autoreactive CTL activation) and at the host level (autoimmune pathologies) by DCs is different and that the hardwired signaling inhibitor prevents DCs from overactivating autoreactive CTLs and causing autoimmune pathologies even when potent inflammatory stimuli are persistently present.
The subsequent mechanistic studies reveal that the inability of matured WT DCs plus persistent TLR signals to break self tolerance at the host level is likely due to the restriction of IL-12 and the downstream cytokine cascade signaling by SOCS1. This conclusion is supported by the following observations: (a) The lack of IL-12 production by SOCS1-silenced DCs (derived from IL-12p35–KO mice) abrogates their ability to break self tolerance at the host level; (b) The blockade of IL-12 signaling in SOCS1-silenced DCs (derived from IL-12Rβ2–KO mice) also abrogates their ability to induce autoimmune pathologies and antitumor activities; (c) SOCS1-silenced DCs produce enhanced levels of IL-12p70 and downstream cytokines with a prolonged duration in response to stimulation; (d) In vivo administration of IL-12 as well as TLR agonists more effectively enhance CTL responses induced by SOCS1-silenced DCs than by WT DCs; and (e) WT DCs transduced with Ad–IL-12 to constitutively overexpress IL-12 are unable to induce autoimmune pathologies.
Maturation is known as the control point for DC transition from the immature, tolerogenic state to the activated, immunogenic state (4, 5). Our results demonstrate that proinflammatory cytokine signaling in mature DCs, restricted by SOCS1, critically regulates the magnitude of CTL responses. This indicates that CTL responses are controlled by DCs on at least 2 levels: the well-defined DC maturation required for the initiation of CTL responses and the SOCS1-restricted cytokine signaling of matured DCs for the control of the magnitude and overactivation of CTL responses uncovered in this study. Our results also imply dynamic interactions between DCs and their surrounding environment of various immune cells and stimuli, which collectively determines the magnitude of CTL responses. It has recently been demonstrated that mature DC lifespan is much longer than previously estimated, lasting 2–3 weeks in vivo (39, 40), supporting the necessity of regulating antigen-presenting DCs after maturation.
This study establishes a new principle for breaking self tolerance and inducing pathological autoimmune responses, which is the first requirement for an effective tumor vaccine, by DCs via silencing a signaling inhibitor. The results of this study suggest the requirement of persistent antigen presentation by DCs for inducing autoimmune pathologies and effective antitumor immunity. Importantly, persistent antigen presentation by DCs cannot be achieved by persistent stimulation with proinflammatory stimuli or overproduction of a key proinflammatory cytokine (IL-12), probably because the hardwired signaling inhibitor prevents the establishment of an intricate autocrine/paracrine cytokine signaling network in DCs and permits DCs to stimulate CTLs only in a transient or pulsing fashion, thus limiting pathological autoimmune responses. The requirement of silencing a signaling inhibitor in order to achieve persistent antigen presentation by DCs for inducing autoimmune pathologies is in concept consistent with earlier findings that vaccination with GM-CSF–producing B16 melanoma cells caused vitiligo only when coinjected with anti-CTLA4 blocking antibodies for inhibiting the negative regulator on T cells (28, 41). However, the requirement for breaking tolerance in transgenic mouse models seems less stringent (29, 42). For example, Yang et al. recently showed that persistent in vivo TLR stimulation of WT DCs could break self tolerance at the host level in a transgenic viral antigen model. However, the conclusion drawn from the transgenic mouse models (29, 42) is not supported by the fact that autoimmune diseases are rare even in individuals with chronic infection with pathogens producing various TLR ligands.
Various inhibitory mechanisms exist in T cells to prevent pathological autoimmunity (26, 43, 44). Disabling of 1 of these inhibitory mechanisms, such as the blockade of CTLA4 on T cells or depletion of CD25+ regulatory T cells, is sufficient to break self tolerance at the host level but causes unwanted nonspecific autoimmune pathologies (26, 45, 46). However, inhibition of a signaling inhibitor in antigen-loaded DCs can induce a TAA-specific, pathological autoimmune response against tumor. The results of our study also suggest a new avenue to improve tumor immunotherapy in general by coadministration of an inhibitor of these signaling inhibitors and a cytokine or TLR agonist.
DCs activated by TLR signaling produce a large number of cytokines, including IL-12, TNF-α, IL-6, IFN-α/β, and IFN-γ, most of which are regulated by SOCS1 (17, 47). The importance of cytokines as a third signal for the activation or overactivation of T cells by DCs has been recognized (12, 13, 48). Production and signaling of proinflammatory cytokines such as IL-12 by WT DCs after TLR signaling is transient (31). Recent studies also showed that autocrine and paracrine signaling of cytokines produced by DCs plays a role in determining the outcome of antigen presentation (30, 49). Here we find that unbridled IL-12 and the downstream cytokine signaling play a critical role in the breaking of tolerance and increased CTL responses. Interestingly, SOCS1–/– inflammatory disease is primarily caused by unbridled IFN-γ signaling (17), although IL-12 also plays a role (47), while LPS-induced toxicity in SOCS1–/– mice is linked to unbridled IFN-α/β signaling (32). Hanada et al. recently reported that SOCS1-KO DCs induced more effective antitumor CTL responses probably due to the enhanced expression of IFN-γ (21). Apparently, other cytokines such as IFN-γ (Figure 7), in addition to IL-12, also play a role in the increased CTL responses induced by SOCS1-silenced DCs. Thus, further studies are warranted to delineate the cellular and molecular details of this SOCS1-regulated, complicated autocrine and paracrine cytokine signaling network in controlling the ability of DCs to activate or overactivate autoreactive CTLs in a natural, suppressive environment. In summary, this study contributes to the fundamental understanding of molecular and immunologic regulation of DCs and may lay a foundation for developing more effective tumor vaccines that not only activate autoreactive T cells, but also cause pathological autoimmune antitumor responses.
Mice. Approval for performing these mouse experiments was obtained from the institutional review board at Baylor College of Medicine. Four- to 6-week-old female C57BL/6, CD4-KO, CD8-KO, IL-12p35–KO, IFN-γ–KO, and IL-12Rβ2–KO mice were purchased from The Jackson Laboratory and maintained in a pathogen-free mouse facility at Baylor College of Medicine according to institutional guidelines.
Peptides. H2-Kb–restricted TRP2 peptides, TRP2a (VYDFFVWL) and TRP2b (SVYDFFVWL) (28), were used for this study. The control H2-Kb–restricted peptide was OVA-I (SIINFEKL). All peptides were synthesized and purified by HPLC to greater than 95% purity by Genemed Synthesis Inc. All peptides were dissolved in DMSO before final dilution in endotoxin-free PBS.
Transduction of BM-derived DCs with lentiviral and adenoviral vectors. Recombinant lentiviral vectors (LV-SOCS1-siRNA and LV-GFP-siRNA) were produced and titrated as described previously (20). A recombinant adenovirus engineered to constitutively express biologically active IL-12 was purchased from InvivoGen and produced according to the manufacturer's instructions. Mouse BM-derived DCs were prepared as described previously (20). Briefly, mouse BM was flushed from the hind limbs, passed through a nylon mesh, and depleted of red cells with ammonium chloride. After extensive washing with RPMI-1640, cells were cultured with RPMI-1640 supplemented with 10% FBS, recombinant mouse GM-CSF/ml (20 ng/ml; PeproTech), and recombinant mouse IL-4 (20 ng/ml; PeproTech). On days 2 and 4 of culture, the supernatant was removed and replaced with fresh medium containing mGM-CSF and mIL-4. All cultures were incubated at 37°C in 5% humidified CO2. Nonadherent granulocytes were removed after 48 hours of culture, and fresh medium was added. After 7 days of culture, more than 80% of the cells expressed characteristic DC-specific markers as determined by FACS. Transductions of mouse BM-derived DCs (day 5–7 of culture) were performed on 12-well plates with addition of 8 μg/ml Polybrene (Sigma-Aldrich) only for lentivirus-mediated transduction. DCs were washed and plated in 12-well plates at a concentration of 1 × 106 cells/well in 400 μl of RPMI-1640. The cells were exposed to lentiviral or adenoviral vectors with different MOIs. After 8–12 hours of transduction, the cells were washed with PBS and further incubated in fresh tissue culture medium supplemented with GM-CSF and IL-4 as described previously (20, 50).
Cytokine ELISA analysis. Levels of various cytokines (IL-12p40, IL-12p70, IFN-γ) were quantitated using the supernatant of DC cultures using ELISA analysis (BD Biosciences) according to the manufacturer's instructions at the time points and with the stimulus as indicated in the figures.
RT-PCR. Total cellular RNA was isolated from DCs using RNeasy (QIAGEN) according to the manufacturer's instructions. The expression level of GAPDH was first evaluated as an internal control using serially diluted reverse-transcribed cDNA. The expression levels of the target mRNAs were then assessed using appropriate pairs of sense and antisense primers: GAPDH, 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′; IL-12p35, 5′-AAATGAAGCTCTGCATCCTGC-3′ and 5′-TCACCCTGTTGATGGTCACG-3′; IL-2, 5′-CTTCAAGCTCCACTTCAAGCT-3′ and 5′-CCATCTCCTCAGAAAGTCCACC-3′; IFN-γ, 5′-TCAAGTGGCATAGATGTGGAAGAA-3′ and 5′-TGGCTCTGCAGGATTTTCATG-3′; eYFP, 5′-CACAAGTTCAGCGTGTCCGGC-3′ and 5′-TCCAGCAGGACCATGTGATGC-3′; IRF-1, 5′-CCTGATGACCACAGCAGTTAC-3′ and 5′-CTTCATCTCCGTGAAGACATG-3′; ICSBP, 5′-GATCAAGGAACCTTCTGTGG-3′ and 5′-GAAGCTGATGACCATCTGGG-3′; c-Rel, 5′-TGGCTGACTGACTCACTGACTGACTGACTCGTGCCTTGC-3′ and 5′-CCAACTAAATCATGAGGATGAGGCTTATATGGATCATTC-3′; C/EBP-β, 5′-GCGGCGAGCGCAACAACATCT-3′ and 5′-TGCTTGAACAAGTTCCGCAG-3′. For analysis of in vivo eYFP-expressing DC lifespan and IL-12p35, CD11c+ cells were first purified from the draining LNs using MACS (Miltenyi Biotec). The PCR products were separated by 1% agarose gel electrophoresis and visualized by ethidium bromide staining.
51Cr CTL assays. CD8+ CTL responses were assessed with a standard chromium release assay, which measures the ability of in vitro–restimulated splenocytes to lyse target cells (20, 50). Splenocytes pooled from immunized mice were restimulated in vitro in RPMI-1640 containing H2-Kb/TRP2 peptide for 4–6 days. TRP2+ target B16 cells, which express TRP2, and control EG.7 cells were labeled with sodium 51Cr chromate solution for 90 minutes at 37°C with shaking. Different numbers of effector cells were incubated with a constant number of target cells (5 × 104/well) in 96-well U-bottomed plates (200 μl/well) for 4 hours at 37°C. The supernatants from triplicate cultures were collected. Percent lysis was calculated as (experimental release – spontaneous release) / (maximum release – spontaneous release) × 100.
IFN-γ ELISPOT assay. ELISPOT assays of isolated CD8+ T cells were performed as described in our previous studies (20). H2-Kb/TRP2 peptide was used for CD8+ T cell stimulation. Irrelevant peptide from OVA was also used as a negative control. Splenocytes or CD8+ cells were isolated from splenocytes using MACS CD8 (Ly-2) MicroBeads (Miltenyi Biotec). Ninety-six-well nitrocellulose-base plates (MultiScreen PI; Millipore) were coated overnight with an anti-murine IFN-γ mAb (Mab AN 18, 10 μg/ml; Mabtech). The plates were washed 6 times with PBS and blocked with RPMI-1640 containing 10% FBS at 37°C for 2 hours. The splenocytes or CD8+ cells, followed by irradiated, peptide-pulsed BM-derived DCs, were seeded into wells and incubated for 20 hours at 37°C in 5% CO2. The cells were then removed by 6 washes with PBS 0.5% Tween-20 (Sigma-Aldrich). Biotinylated anti-mouse IFN-γ antibody (Mab R4-6A2 biotin; Mabtech), diluted in PBS containing 0.5% FBS to 1 μg/ml, was added, and the mixture incubated at 25°C for 2 hours. The avidin/biotinylated enzyme complex (ABC; Vector Laboratories) was added for an additional hour. Cytokine-secreting cells were detected after a 4 minute reaction with AEC (3-amino-9-ethylcarbazole; Sigma-Aldrich). The results were evaluated by ZellNet Consulting Inc. with an automated ELISPOT reader system (Zeiss), using KS ELISPOT 4.3 software.
Tetramer staining. H2-Kb/TRP2 tetramer assays were used to detect TRP2-specific CD8+ T cells. TRP2 tetramers were synthesized at the Baylor College of Medicine Tetramer Core Facility. Splenocytes from immunized mice were double stained with anti–CD8α-FITC and H2-Kb/TRP2-PE tetramers on different days after DC immunization. Tetramer staining was done at 4°C, for 1 hour with 1 μg of anti-CD8α and a 1:100 dilution of TRP2 tetramers per 106 cells, according to the manufacturer's instructions.
Intracellular cytokine staining. Splenocytes were harvested from immunized mice and cultured with TRP2 peptide for 8 hours at 37°C. For the final 6 hours of culture, GolgiPlug (BD Biosciences — Pharmingen) was added to the supernatant. After surface staining with anti-CD8, cells were permeabilized and stained for intracellular IFN-γ, with anti–IFN-γ (BD) as we have previously described (51).
Western blotting. SOCS1-siRNA DCs or GFP-siRNA DCs were continuously stimulated with LPS (Sigma-Aldrich) and plate-immobilized anti-CD40 mAb (BD Biosciences — Pharmingen). Twenty-four, 48, or 72 hours later, the cells were harvested and subjected to SDS-PAGE (20). Following transfer to Hybond-P membrane (Amersham Biosciences), the samples were analyzed by Western blotting with monoclonal anti-pStat4 (Santa Cruz Biotechnology Inc.) or polyclonal anti-Stat4 (Santa Cruz Biotechnology Inc.) antibodies, followed by detection with ECL-Plus reagent (Amersham Biosciences).
DC transfer and tumor mouse study. BM-derived DCs (day 4–5 of BM culture) were transduced with LV-SOCS1-siRNA or LV-GFP-siRNA at an MOI of 5 or Ad-IL-12 vector at an MOI of 300 or 1,000 (20). DCs were then pulsed with TRP2 peptides for 20 hours, washed with PBS 3 times, stimulated with LPS (100 ng/ml; Sigma-Aldrich) or TNF-α (50 ng/ml; R&D Systems) for 24 hours, washed with PBS 3 times, and then injected into C57BL/6 mice via a rear foot pad. In the therapeutic model, B16 tumor cells (2.5 × 105) were injected s.c. into the right flank of syngeneic mice to establish a tumor model. On day 3 after tumor inoculation, the mice were randomly divided into groups and injected with antigen-pulsed, matured, transduced DCs (1.5 × 106) or PBS control. In some mice, TLR agonist (LPS [30 μg/mouse; Sigma-Aldrich], poly[I:C] [50 μg/mouse; InvivoGen], peptidoglycan [25 μg/mouse; InvivoGen], imiquimod [50 μg/mouse; InvivoGen]) or IL-12 (1 μg/mouse; PeproTech) was administered i.p. after DC vaccination on the days indicated in the figures. Tumor volumes were measured every 2–4 days with an electronic caliper until the experiment was completed.
Statistics. For statistical analysis, we used 2-tailed Student's t test, and a 95% confidence limit was taken to be significant, defined as P < 0.05. Results are presented as mean ± SEM.
We thank Lisa Rollins, Wenhong Ren, Natasha Lapteva, Andrew Sharabi, Jiali Sun, and Lei Shen for technical assistance and valuable suggestions. This work was supported by grants from the NIH (R01CA90427, R0148480, and R01AI48711), the Leukemia and Lymphoma Society SCOR, and the US Army Prostate Cancer Research Program (to X.F. Huang). K. Evel-Kabler and M. Aldrich were supported by a predoctoral and postdoctoral NIH training grant (T32-AI07495).
Nonstandard abbreviations used: ELISPOT, enzyme-linked immunospot; TAA, tumor-associated antigen; TRP2, tyrosinase-related protein 2.
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Q: Trying to make post request to spring controller I am trying to make a post request with jquery to a spring rest controller I have setup. This works perfectly fine with the get request, however the post request is giving me a 403 error in the console. Here is my simple code.

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<script src="https://ajax.googleapis.com/ajax/libs/jquery/2.1.1/jquery.min.js"></script>

And my controller
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redpajama_set_name: RedPajamaStackExchange


text:
Why Single Payer?
How Much is ObamaCare Penalty?
A lot of Americans were not really happy upon knowing that the ObamaCare now requires them to purchase health care plans. Many fail to see the importance of a health care plan in their life and this is what ObamaCare emphasizes. So, since the ObamaCare Tax Penalty is now inevitable, a ray of light can still be seen in this case. The ObamaCare Tax Penalty for people who do not want to purchase health care plans is lesser than an actual health insurance. But how much is the ObamaCare Tax Penalty?
There is no fixed amount for everyone else to pay if they opt not to purchase any health care plan. The amount will be dependent on some factors. Here are several key points that you should know about the penalty for not having insurance.
In 2014, the minimum tax or penalty per person will fall at $95 and then it will gradually increase through to 2016.
In 2016, the minimum tax or penalty per person will be at $695 and it can be up to three times of that per household or family. After the year 2016, these amounts will increase depending on the inflation rate.
Regardless of the number of people in the family, payment will not go over 3x of the per person tax penalty.
The penalty of $695 per person will only apply to those who earn between $9500 up to $37000 per annum. If you are making less than the minimum, you will be exempted. If you are earning more than the max range, the penalty will be calculated based on a formula.
The formula will be 2.5% of the household income that fall above the level in which you will be required to file for an ITR or Income Tax Return. So, if you are earning more, the penalty from your taxable income will also be more. On the lighter note, the ObamaCare Tax Penalty cannot go beyond the Bronze Health Plan.
Here is a list of the possible ObamaCare Tax Penalty depending on the income level per annum:
$9499 or less income equals $0 tax penalty
$9500 up to $37000 income equals $695 tax penalty
$50000 income equals $1000 tax penalty
$100000 income equals $2250 tax penalty
$200001 income or more equals the same amount or cost of the Bronze Health Plan as tax penalty
The tax penalty will then be collected by the IRS. There will be exemptions to the ObamaCare Tax Penalty though, and these exemptions include:
Those who are making $9499 and lesser
Those employees who have employers offering plans that amount to 8% or more of the employees' salary
Those who are currently experiencing "hardships"
Those who belong to Indian tribes
Those who are members or certain religions that are not paying any Social Security Tax like the Hutterites, Mennonites, or the Amish.
If you are unsure of how much your ObamaCare Tax Penalty is, you will need to check your income per annum and do the math from there. On the other hand, you can also consult an accountant about it.
See Also Universal Health Care in Different Countries:
Universal Health Care in Canada
Single-Payer Health Care
Universal Health Care in the United Kingdom
French Health Care
Healthcare in Taiwan
Countries with Better Healthcare than the United States
Pros and Cons of ObamaCare
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Paytm employees became millionaires overnight! Here's how
Do you have your ESOPs in place?
Paytm revealed on Monday that it had reached a valuation of $10 billion. The rise in value came after about 200 employees, current and former, liquidated a portion of the Paytm shares they held as part of their employee stock ownership plan (ESOP) for an estimated value of INR300 crore, making them millionaires almost overnight. Employee stock benefit plans are offered by companies (particularly startups) as a benefit to employees in order to improve the company's performance by encouraging the people working in it to take ownership. The ESOP rags-to-riches story began with reports of drivers, plumbers and attendants becoming millionaires at Infosys at the turn of the century. Even today, the trend continues. According to Moneycontrol, the ESOPs in question were sold to unidentified investors, including some foreign funds and family offices. "Paytmers who have been with the company since inception to as early as one year have benefited," according to a statement from Paytm.
But while INR300 crore from about 200 employees' ESOPs surely seems like a huge amount, it is important to note that about a year ago, when Paytm needed funds for its Paytm Payments Bank project, the company's founder and CEO, Vijay Shekhar Sharma, sold a single percent of his share in the company and managed to raise INR325 crore. The e-wallet has seen a massive jump in valuation. A previous estimation showed that Paytm's value stood at $7 billion in May 2017 when it had managed to raise $1.4 billion from Japan's SoftBank.
However, Paytm isn't the only one whose share-holding employees have seen a rise in fortunes. Only last month, Flipkart (valued at $12 billion) repurchased ESOPs worth $100 million. So if you're currently employeed at a startup - and you know it's going places - maybe it's time to talk to your HR about stock options.
Vijay Shekhar Sharma is India's youngest billionaire
Vijay Shekhar Sharma's famous, super rich neighbours in Lutyens' Delhi
Vijay Shekhar Sharma is probably the luckiest man in India right now
KeywordsBENEFITDELHIFlipkartIndiaInfosys
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Warren Buffett: Don't Invest in S&P 500 Index Fund 2023 (WHY?)
In this article, I will show how some direct Warren Buffett quotes from a recent question could apply to investors for not buying the S&P 500 index fund in 2023 using his parameters.
In recent years at a Berkshire Hathaway shareholder meeting, a lady asked a well-thought-out question to, Warren Buffett. Suppose he believes so much in buy-and-hold investing, specifically in the S&P 500 index. Why doesn't he hold Berkshire's excess cash above the cash needed for insurance underwriting in equities? This would have led to a huge outperformance above his already great returns for the Berkshire portfolio.
Here is her question:
"Warren, you are a big advocate of index investing and of not trying to time the market, but by your having Berkshire hold such a large amount of cash and T-bills, it seems to me you don't practice what you preach. I'm thinking that a good alternative would be for you to invest most of Berkshire's excess cash in a well-diversified index fund until you find an attractive acquisition or buyback stock." she continues, "Had you done that over the past 15 years, all the time keeping the $20 billion cash cushion you want, I estimate that at the end of 2018, the company's $112 billion balance in cash, cash equivalents, and short term investments in T-bills would have
instead been worth about $155 billion. The difference between the two figures is an opportunity cost equal to more than 12 percent of Berkshire's current book value."
This is how Warren Buffett responded to her question about keeping all his excess cash in index funds instead of treasury bills:
"That's a perfectly decent question, and I wouldn't quarrel with the numbers, and I would say that is an alternative, for example, that my successor may wish to employ. Because on balance, I would rather own an index fund than carry treasury bills. I would say that if we'd instituted that policy in 2007 or eight, we might have been in a different position in terms of our ability to move in late 2008 or 2009. It has certain execution problems with hundreds of billions of dollars than it does if you were having a similar policy with a billion or two billion or something sort, but it's perfectly rational observation, and certainly, looking back on ten years of a bull market it really jumps out at you, but I would argue that if you were working with smaller numbers, it would make a lot of sense, and if
they're working with large numbers it might well make sense in the future. Berkshire would operate that way, you know, we committed $10 billion a week ago, and there are conditions under which and they're not they're not remote, they're not likely in any given week or month or year, but there are conditions under which we could spend $100 billion dollars very very quickly, and if we did if those conditions existed it would be the capital very well deployed and much better than in an index fund so we've been uh we're operating on the basis that we will get chances to deploy capital they will come in clumps in all likelihood and they will come when other people
don't want to allocate capital, Charlie; what do you think about it?"
Charlie Munger continued the response to her question following Buffett's points:
"Well, I plead guilty to being a little more conservative with the cash than other people and but I think that's all right. We could have put all the money into a lot of securities that would have done better than the S&P with 2020 hindsight. Remember we had all that extra cash all that period if something had come along in the way of opportunities and so on. I don't think it's a sin to be a little strong on cash when you're as big a company as we are. We don't have to. I watched Harvard use the last ounce of their cash including all their prepaid tuition from the parents and plunge it into the market at exactly the wrong moment and make a lot of forward commitments to private equity, and they suffered like two or three years of absolute agony we don't want to be like Harvard. Because of timber and a whole bunch of things. We're not going to change."
Warren Buffet interjected:
"We do like having a lot of money to be able to operate very fast and very big. You know, and maybe we won't, we know we won't get those opportunities frequently I don't think, certainly you know, in the next you know, in the next 20 or 30 years, they'll be good two or three times when it'll be raining gold and all you have to do is go outside but uh we don't know when they will happen and we have a lot of money to commit and I would say that if you told me I had to either carry short-term treasury bills or have index funds and just let that money be invested in America generally, I would take the index funds, but we still have hopes, and one thing you should very definitely understand about Berkshire is that we run the business in a way that we think is consistent with serving shareholders who have virtually all of their net worth in Berkshire. I happen to be in that position myself, but I would do it that way under any circumstances we have a lot of people who trust us who really have disproportionate amounts of Berkshire compared to their net worth. If you were to follow standard investment procedures and we want to make money for everybody, but we want to make very, very sure that we don't lose permanently money from anybody for anybody that buys our stock somewhere around intrinsic business value, to begin with. We just have enough, we have an aversion to having million-plus shareholders, maybe as many as two million, and having a lot of them ever really lose money if they're willing to stay with us for a while, and we know how people behave if when the world generally is upset, and they want to be with something I think they want to be with something that they feel was like the rocketed roller, and we have a real disposition toward that group."
Here are some interesting points to take away from his response:
It's good to have a large cash reserve to buy great individual stocks at a value when the opportunity arises.
It's good to raise cash during high-valuation bull markets like 2007 to buy during bear market crashes.
Buffett advises only buying even Berkshire stock at fair value to optimize future returns.
Buying and holding only looks like a great strategy in hindsight and after long bull markets.
Due to the risk, Buffett did not want to hold all his cash in stock index funds in late 2007.
Buffett does not say anything about dollar cost averaging but looks to buy during great opportunities with low stock valuations.
For investors who think stock valuations are still too high in 2023 and the stock market has lower to go based on the economy, Buffett's advice could mean to raise cash and wait for the stocks you want and the S&P 500 index to return to a more fair value.
"It's far better to buy a wonderful company at a fair price, than a fair company at a wonderful price." – Warren Buffett
Video source here.
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In May of 2013, a half-mile wide tornado roared through the Plaza Towers Elementary School in Moore, Okla. Its 200-mph winds tore the school and the surrounding community to shreds.
Like fires and some other kinds of natural disasters, tornados come out of nowhere threatening and often taking lives. Brutal winds ranging from 40 mph to over 300 mph will not only kill people, they will destroy property. Raging winds can pick up cars, as well as people, and toss them around like a child's toys.
Hurricanes wreak havoc on lives and properties, too, but weather forecasters can see hurricanes coming and predict their paths with reasonable accuracy, allowing people at least a couple days to prepare.
In fact, it is a good idea for K-12 schools to be prepared well ahead of any kind of disaster. Then when something happens, administrators and students can take cover immediately in a shelter strong enough to protect lives for days. Preparing for natural disasters involves identifying risks and finding ways to remove their sting. Preparations also include readying shelters, warning everyone with alarms and communications, setting up a system that will enable a school to get back to teaching as quickly as possible.
"Look at your region of the country and identify the kinds of disasters that you might face," says Michael Dorn, executive director of the Juliette, Ga.-based Safe Havens International, the world's largest school safety center. "The two biggest risks are earthquakes and tornados. Don't ignore events because they are infrequent. For instance, earthquakes are not common on the east coast, but they have occurred there and they will again.
Take some time to make sure the shelters chosen will provide adequate protection. Some schools have been getting it wrong.
"The way many schools have drilled to prepare for tornados is wrong," says Paul Timm, vice president of RETA Security, a division of Fairfax, Va.-based Facility Engineering Associates. "They prepare by sending students into the school's hallways because the walls there are reinforced.
Where can people shelter safely? The answer, says Timm, is the basement, if the facility has one. If not, move into the interior rooms, rooms with no windows — in a school, interior rooms typically include restrooms and locker rooms. Timm also notes that K-12 schools are required to post evacuation maps that teachers and students can refer to in the event of fire. "Our company also recommends that these maps address severe weather by designating shelter areas," he says.
According to the 2015 International Building Codes, all new schools must have tornado shelters built to FEMA specifications. While the 2015 Codes are a couple years old, architects say that many schools have not yet been brought into compliance with these requirements.
Students, teachers and people from the local community may use schools as shelter for a different kinds of disasters: hurricanes, floods, wildfires and so on. With that in mind, Timm suggests keeping emergency supplies on hand in schools. "We recommend Classroom Emergency Backpacks," he says. "These backpacks have supplies of food — specifically hypo-allergenic energy bars — and water. They also have wind-up flashlights, which don't need batteries, thin, folded Mylar blankets, glow sticks, and first aid kits with emergency medical supplies.
Don't forget tents, continues Timm. Suppose an event destroys a wing of the building with several classrooms. Tents can provide temporary classrooms until building repairs are complete.
In addition to emergency supplies, it is important to drill for natural disasters. While that may sound like too much trouble, experts point out that K-12 schools have been drilling for fire emergencies for decades now. And when there is a fire, students as well as teachers have proven adept at getting out of the school to safety.
"Drills are important," says Timm. "In our state, Illinois, all public and private K-12 schools are required to have one severe weather emergency drill every year — in addition to fire evacuation drills, lockdown drills and other drills that might be recommended.
Not all states mandate drills, but Timm believes drilling is key to surviving a disaster relatively unscathed.
Timm also helps schools organize emergency table-top exercises for various scenarios such as severe weather. Table-top drills bring school emergency managers, administrators, police and firefighters together to discuss what they would do to manage various emergency scenarios. Another way table-top drills help is that everyone at the table sees what each of the others is going — so participants get more than just their own angles.
One more option that some districts have adopted is a short video, about two minutes, that explains what to do during an evacuation. Such videos are particularly useful when regular drills are impractical.
On March 2, 2012, a tornado caused significant damage to the Henryville High School in Henryville, Ind. Students, who were sheltering in place inside the school, survived with nothing more serious than minor cuts and bruises.
Fires and sudden severe stores require quick action by everyone. School officials must notify students and teachers and spur an immediate reaction.
"Everything today is mass notification," says Timm. "That means I need several communication vehicles. A midwestern town, for instance, may have a tornado siren, while buildings may notify occupants through their public-address systems.
Some schools are installing emergency message boards. Information about a threat can be programmed to scroll across these boards. If conditions are right to produce a tornado, for instance, the boards on campus might say: Tornado watch. Please move to a safe area.
"These messages must get through to everyone," Timm says. "So you should use as many ways to communicate as you have. Other communication methods in use today include texting and emailing to cell phones, tablets and computers.
Getting through a natural disaster with as few injuries and as little property damage as possible is one part of emergency management. Once the emergency ends, a school must get back to teaching students. Dorn calls this the Continuity Of Operations Plan (COOP).
"Say an earthquake causes extensive damage to a school," Dorn explains. "The emergency plan helps to keep people alive. Next comes COOP. Suppose inspections show that part of a building — or an entire building — is out of commission. A detailed COOP plan will address a variety of physical and IT scenarios and problems with solutions designed to return buildings to service as quickly as possible.
Preparing for natural disasters ranks as a vitally important task for school administrators and staff. It requires taking stock of risks facing a school in the event of a flood, earthquake, fire or any other natural disaster that might befall a school.
The preparatory thinking and work is often assigned to the security director and his or her staff. Experts recommend that the security director lead the effort by eliciting broad participation from the administration, faculty, student body and parents. In the end, disaster preparations shared across the community will prove to be highly effective when the time comes to make use of them.
When students, faculty and administrators evacuate a school, just about everyone stands up and walks in orderly lines out of the school, just as they did during regular drills.
Then again, not everyone can stand up and walk out. What about people with disabilities? People in wheelchairs or on crutches can't simply walk out, and safety professionals ask that elevators not be used in an emergency. People with disabilities need special help.
"Lift and carry can be dangerous," says Jeff Wolvovsky of EVAC+CHAIR North America LLC in Lake Success, N.Y.


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#1 is the Parotid gland; #2 is the Submandibular gland; #3 is the Sublingual gland.
The salivary glands make saliva. Saliva keeps the mouth and other parts of the digestive system wet and slippery. They also help break down the food when you are chewing. This helps food go down the throat to the stomach.
There are also many small glands in the tongue, cheeks, lips and palate. All of those glands make mucous.
Saliva consists of two fluids, mucous and serous.
Mucous (slime) is a saccharide glycoprotein called mucin. With water it makes the slippery lubricant used in many parts of the body, where it lines the moving parts, surfaces and tubes. Here it lubricates the mouth, throat and alimentary canal.
The mucous also has antiseptic qualities: it contains lysozyme and immunoglobulins. Mucous, with its antiseptic molecules and slime, traps fungi, bacteria and viruses and prevent infections. The body produces about a litre of mucous per day, in the mouth and other places.
The serous fluid contains the enzyme amylase which acts in the digestion of carbohydrates. Minor salivary glands on the tongue secrete the amylase. The parotid gland produces purely serous saliva. The other major salivary glands produce mixed (serous and mucus) saliva.
Another type of serous fluid is secreted by the two layered serous membranes which line the body cavities. The serous fluid between the two layers acts as a lubricant and reduces friction from muscle movement.
The parotid gland makes serous secretions only.
The submandibular glands are mixed glands. They give serous and mucous secretions.
The sublingual glands make mucous only.
Serous cells are shaped like a pyramid. They are joined together in a group that is shaped like a ball.
Mucous cells are usually shaped like a cube. They are joined together to make a tubules, which are very small tubes.
Myoepithelial cells are all around the part of the gland where the saliva comes out. They can squeeze the saliva gland so the saliva comes out faster.
The small tubes go into ducts. Those ducts go into larger ducts that have little stripes on them, called striations. Those go into ducts between the lobes of the gland (called interlobar or excretory ducts). The main duct of the salivary glands then opens into the mouth.
The two parotid glands are near the ear (par- = next to, -otid = ear). They are the largest salivary glands.
The sublingual gland is under the tongue.
The submandibular gland is in the shape of a "U". It is under the chin bone which is called the mandible.
Ducts can get blocked. This would cause pain and swelling of the gland.
Tumours of the salivary glands can occur. These are usually benign. Investigation is done by a sialogram, which is a radiocontrast study of a salivary duct.
This page was last changed on 3 April 2018, at 23:11.


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At Harner Orthodontics, we strive to exceed your expectations by providing you and your family with personalized attention, and an exceptional quality of care to make the process of creating a beautiful new smile as easy and convenient as possible. Dr. Harner and our expert team of highly trained orthodontic specialists work hard to get to know all of our patients individually, designing personalized treatment plans that perfectly suit each patient's unique orthodontic needs. With a passion for excellence, our mission is to provide the highest standard in orthodontic care using the latest techniques and state-of-the-art technology in a warm, welcoming, and positive office environment.
If you need to reach us after hours, you can leave a message on our answering service and we'll return your call as soon as possible.
We work hard to accommodate the schedules of our patients, so we ask that you remember that your scheduled appointment has been reserved specifically for you. To ensure we provide the best care to all our patients, we ask for 24-hour notice if you need to cancel an appointment. That said, we know that sometimes things come up - but after missing your second appointment without giving us 24-hours notice, you'll be subject to an additional fee.
If you have more questions about our office or would like to come in for a visit, we'd be more than happy to speak with you. Feel free to call us at (714) 842-9933, reach out to us at our contact page, or use our easy online form to schedule an appointment at our Huntington Beach, CA office. We can't wait to hear from you, and we look forward to helping you achieve a beautiful, healthy new smile that'll last you for years to come!


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Q: Does MongoClient.getDatabase() return just a DB instance or actually creates it? In order to create a database instance in MongoDB 3.2 you have to write something like that:
new MongoClient().getDatabase("dbName");

According to the official MongoDB Java API 3.2 this should return you a database instance.
My questions:

*

*Do I understand correctly, that if the DB with the specified name does not exist, thus MongoDB will create it for me?

*And if the assumption from the question #1 is correct, what will happen if I'll try to request the same DB twice? Will it return just the already existed instance or will drop the existed one and will create a new one?
P.S. Can you, please, advise me a good and updated MongoDB for Java manual/tutorial, I googled for a couple of them, but they are not so updated.

A: In 3.1.1 driver:
Calling getDatabase doesn't in fact create new database because operation is lazy - it returns database representation. Calling any modifiable operation (e.g. createCollection):

*

*will create new database for you if it is not present

*if present it will get database for you

But remember that you have to call any operation which actually performs something - like create. If you just call getDatabase it won't create it.