Induction Of CTL Responses Research Articles

Immunoinformatics approach for designing a multi-epitope subunit vaccine against porcine epidemic diarrhea virus genotype IIA spike protein

Background: Porcine epidemic diarrhea (PED), caused by the porcine epidemic diarrhea virus (PEDV), is associated with high mortality and morbidity rates, especially in neonatal pigs. This has resulted in significant economic losses for the pig industry. PEDV genotype II-based vaccines were found to confer better immunity against both heterologous and homologous challenges; specifically, spike (S) proteins, which are known to play a significant role during infection, are ideal for vaccine development. Aim: This study aims to design a multi-epitope subunit vaccine targeting the S protein of the PEDV GIIa strain using an immunoinformatics approach for the development of a multi-epitope subunit vaccine targeting the S protein of PEDV GIIa strain. Methods: Various bioinformatics tools were used to predict HTL, CTL, and B-cell epitopes. The epitopes were connected using appropriate linkers and conjugated with the CTB adjuvant and M-ligand. The final multi-epitope vaccine construct (fMEVc) was then docked to toll-like receptor 4 (TLR4). The stability of the fMEVc - TLR4 complex was then simulated using GROMACS. C-immsim was then used to predict the in vitro immune response of the fMEVc. Results: Using various bioinformatics tools, six (6) epitopes were predicted to induce antibody production, ten (10) epitopes were predicted to induce CTL responses, and four (4) epitopes were predicted to induce HTL responses. The assembled epitopes conjugated with the CTB adjuvant and M-ligand, fMEVc, is antigenic, non-allergenic, stable, and soluble. The construct showed a favorable binding affinity for TLR4, and the protein complex was shown to be stable through molecular dynamics simulations. A robust immune response was induced after immunization, as demonstrated through immune stimulation. Conclusion: In conclusion, the multi-epitope subunit vaccine construct for PEDV designed in this study exhibits promising antigenicity, stability, and immunogenicity, eliciting robust immune responses and suggesting its potential as a candidate for further vaccine development.

Sec22b and Stx4 Depletion Has No Major Effect on Cross-Presentation of PLGA Microsphere-Encapsulated Antigen and a Synthetic Long Peptide InVitro.

The induction of CTL responses by vaccines is important to combat infectious diseases and cancer. Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres and synthetic long peptides are efficiently internalized by professional APCs and prime CTL responses after cross-presentation of Ags on MHC class I molecules. Specifically, they mainly use the cytosolic pathway of cross-presentation that requires endosomal escape, proteasomal processing, and subsequent MHC class I loading of Ags in the endoplasmic reticulum (ER) and/or the endosome. The vesicle SNARE protein Sec22b has been described as important for this pathway by mediating vesical trafficking for the delivery of ER-derived proteins to the endosome. As this function has also been challenged, we investigated the role of Sec22b in cross-presentation of the PLGA microsphere-encapsulated model Ag OVA and a related synthetic long peptide. Using CRISPR/Cas9-mediated genome editing, we generated Sec22b knockouts in two murine C57BL/6-derived APC lines and found no evidence for an essential role of Sec22b. Although pending experimental evidence, the target SNARE protein syntaxin 4 (Stx4) has been suggested to promote cross-presentation by interacting with Sec22b for the fusion of ER-derived vesicles with the endosome. In the current study, we show that, similar to Sec22b, Stx4 knockout in murine APCs had very limited effects on cross-presentation under the conditions tested. This study contributes to characterizing cross-presentation of two promising Ag delivery systems and adds to the discussion about the role of Sec22b/Stx4 in related pathways. Our data point toward SNARE protein redundancy in the cytosolic pathway of cross-presentation.

Identification of HLA-A2 restricted epitopes of glypican-3 and induction of CTL responses in HLA-A2 transgenic mice.

Hepatocellular carcinoma (HCC) is a malignant tumor with high mortality, but lacks effective treatments. Carcinoembryonic antigen glypican-3 (GPC3) is a tumor-associated antigen overexpressed in HCC but rarely expressed in healthy individuals and thus is one of the most promising therapeutic targets. T cell epitope-based vaccines may bring light to HCC patients, especially to the patients at a late stage. However, few epitopes from GPC3 were identified to date, which limited the application of GPC3-derived epitopes in immunotherapy and T cell function detection. In this study, a total of 25 HLA-A0201 restricted GPC3 epitopes were in silico predicted and selected as candidate epitopes. Then, HLA-A0201+/GPC3+ HCC patients' PBMCs were collected and co-stimulated with the candidate epitope peptides in ex vivo IFN-γ Elispot assay, by which five epitopes were identified as real-world epitopes. Their capacity to elicit specific CD8+ T cells activation and proliferation was further confirmed by in vitro co-cultures of patients' PBMCs with peptide, in vitro co-cultures of healthy donors' PBLs with DCs and peptide, T2 cell binding assay as well as HLA-A2 molecule stability assay. Moreover, the in vivo immunogenicity of the five validated epitopes was confirmed by peptides cocktail/poly(I:C) vaccination in HLA-A0201/DR1 transgenic mice. Robust epitope-specific CD8+ T cell responses and cytotoxicity targeting HepG2 cells were observed as detected by IFN-γ Elispot, intracellular IFN-γ staining and cytolysis assay. This study provided novel GPC3 CTL epitopes for the development of T cell epitope vaccines and evaluation of GPC3 specific T cell responses.

Cutting Edge: The RIG-I Ligand 3pRNA Potently Improves CTL Cross-Priming and Facilitates Antiviral Vaccination.

Protective immunity against intracellular pathogens involves the induction of robust CTL responses. Vaccination with protein Ags establishes such responses only when combined with immune-stimulatory adjuvants. In this study, we compared different adjuvants and identified triphosphate RNA (3pRNA) as especially effective at inducing CTL responses. 3pRNA sensing required IPS-1/MAVS signaling and induced type I IFN in plasmacytoid dendritic cells and macrophages, with the latter being more important for the adjuvant effect. Type I IFN acted on CD11c(+) cells, especially on CD8α(+) Batf3-dependent dendritic cells. Vaccination with OVA in combination with 3pRNA protected mice from a subsequent OVA-encoding adenovirus infection in a CD8(+) cell-dependent manner and more efficiently than other adjuvants. In summary, 3pRNA is a superior adjuvant for CTL activation and might be useful to facilitate antiviral immunization strategies.

Mechanism for cytoplasmic transduction peptide-HBcAg18-27-Tapasin to induce HBV specific CTL response in C57BL/6 mice

目的：观察融合蛋白胞质转导肽（cytoplasmic transduction peptide,CTP）-HBcAg_（18-27）-Tapasin通过介导JAK/STAT通路诱导近交系C57BL/6小鼠HBV特异性细胞毒性T淋巴细胞（cytotoxic T lymphocyte,CTL）反应.方法：C57BL/6小鼠随机分为4组：CTPHBcAg_（18-27）-Tapasin实验组、CTP-HBcAg_（18-27）-Tapasin＋AG490对照组、AG490对照组、PBS空白组.融合蛋白经肌肉注射免疫小鼠,腹腔注射AG490阻断JAK/STAT通路,CCK-8比色法检测T淋巴细胞增殖活性;流式细胞术检测T淋巴细胞内的的细胞因子;ELISA检测T淋巴细胞分泌细胞因子,Real-time PCR检测JAK/STAT通路信号分子表达水平.结果：CTP-HBcAg_（18-27）-Tapasin组相比CTPHBcAg_（18-27）-Tapasin＋AG490组CD8~＋IFN-γ~＋T双阳性细胞百分比显著增加（P〈0.01）,淋巴细胞增殖活性差异具有显著性（P〈0.01）,Th1型细胞因子分泌水平显著增加（P〈0.01）,其他各组之间没有显著性差异,JAK/STAT信号通路信号中Jak2,STAT4 mRNA分子表达水平CTP-HBcAg_（18-27）-Tapasin组相比其他对照组表达水平差异具有显著性（P〈0.05）,CTP-HBcAg_（18-27）-Tapasin组Tyk2、STAT1mRNA的分子表达水平相比其他对照组表达水平具有显著性差异（P〈0.01）.结论：本研究表明CTP-HBcAg_（18-27）-Tapasin通过JAK/STAT信号通路促进Th1型细胞因子的分泌而促进特异性CTL反应.

Langerhans Cell-Dendritic Cell Cross-Talk via Langerin and Hyaluronic Acid Mediates Antigen Transfer and Cross-Presentation of HIV-1.

Human epidermal and mucosal Langerhans cells (LCs) express the C-type lectin receptor langerin that functions as a pattern recognition receptor. LCs are among the first immune cells to interact with HIV-1 during sexual transmission. In this study, we demonstrate that langerin not only functions as a pattern recognition receptor but also as an adhesion receptor mediating clustering between LCs and dendritic cells (DCs). Langerin recognized hyaluronic acid on DCs and removal of these carbohydrate structures partially abrogated LC-DC clustering. Because LCs did not cross-present HIV-1-derived Ags to CD8(+) T cells in a cross-presentation model, we investigated whether LCs were able to transfer Ags to DCs. LC-DC clustering led to maturation of DCs and facilitated Ag transfer of HIV-1 to DCs, which subsequently induced activation of CD8(+) cells. The rapid transfer of Ags to DCs, in contrast to productive infection of LCs, suggests that this might be an important mechanism for induction of anti-HIV-1 CD8(+) T cells. Induction of the enzyme hyaluronidase-2 by DC maturation allowed degradation of hyaluronic acid and abrogated LC-DC interactions. Thus, we have identified an important function of langerin in mediating LC-DC clustering, which allows Ag transfer to induce CTL responses to HIV-1. Furthermore, we showed this interaction is mediated by hyaluronidase-2 upregulation after DC maturation. These data underscore the importance of LCs and DCs in orchestrating adaptive immunity to HIV-1. Novel strategies might be developed to harness this mechanism for vaccination.

Rate of pESC plasmid loss in Saccharomyces cerevisiae in the absence of selective pressure (VAC13P.1126)

Abstract Recombinant yeast has been proposed as a vaccine modality, especially for inducing CTL responses. The production of yeast expressing proteins of interest would be facilitated if they can be grown in less expensive non-selective medium. This would only be practical if the population of yeast retains high levels of plasmid over the duration of the growth. We tested the Saccharomyces cerevisiae multiple gene auxotroph with pESC-URA to determine the rate of plasmid loss in the population. The yeast maintain the plasmid much longer than anticipated, suggesting that growth in non-selective medium may be a viable method. Induction of protein expression increases the competitive advantage of plasmid loss. Growth rates of the yeast and of plasmid loss under these variable conditions was also determined.

Glioma-associated antigen HEATR1 induces functional cytotoxic T lymphocytes in patients with glioma.

A2B5+ glioblastoma (GBM) cells have glioma stem-like cell (GSC) properties that are crucial to chemotherapy resistance and GBM relapse. T-cell-based antigens derived from A2B5+ GBM cells provide important information for immunotherapy. Here, we show that HEAT repeat containing 1 (HEATR1) expression in GBM tissues was significantly higher than that in control brain tissues. Furthermore, HEATR1 expression in A2B5+ U87 cells was higher than that in A2B5−U87 cells (P = 0.016). Six peptides of HEATR1 presented by HLA-A∗02 were selected for testing of their ability to induce T-cell responses in patients with GBM. When peripheral blood mononuclear cells from healthy donors (n = 6) and patients with glioma (n = 33) were stimulated with the peptide mixture, eight patients with malignant gliomas had positive reactivity with a significantly increased number of responding T-cells. The peptides HEATR1682–690, HEATR11126–1134, and HEATR1757–765 had high affinity for binding to HLA-A∗02:01 and a strong capacity to induce CTL response. CTLs against HEATR1 peptides were capable of recognizing and lysing GBM cells and GSCs. These data are the first to demonstrate that HEATR1 could induce specific CTL responses targeting both GBM cells and GSCs, implicating that HEATR1 peptide-based immunotherapy could be a novel promising strategy for treating patients with GBM.

Role of Fatty-Acid Synthesis in Dendritic Cell Generation and Function

Dendritic cells (DC) are professional APCs that regulate innate and adaptive immunity. The role of fatty-acid synthesis in DC development and function is uncertain. We found that blockade of fatty-acid synthesis markedly decreases dendropoiesis in the liver and in primary and secondary lymphoid organs in mice. Human DC development from PBMC precursors was also diminished by blockade of fatty-acid synthesis. This was associated with higher rates of apoptosis in precursor cells and increased expression of cleaved caspase-3 and BCL-xL and downregulation of cyclin B1. Further, blockade of fatty-acid synthesis decreased DC expression of MHC class II, ICAM-1, B7-1, and B7-2 but increased their production of selected proinflammatory cytokines including IL-12 and MCP-1. Accordingly, inhibition of fatty-acid synthesis enhanced DC capacity to activate allogeneic as well as Ag-restricted CD4(+) and CD8(+) T cells and induce CTL responses. Further, blockade of fatty-acid synthesis increased DC expression of Notch ligands and enhanced their ability to activate NK cell immune phenotype and IFN-γ production. Because endoplasmic reticulum (ER) stress can augment the immunogenic function of APC, we postulated that this may account for the higher DC immunogenicity. We found that inhibition of fatty-acid synthesis resulted in elevated expression of numerous markers of ER stress in humans and mice and was associated with increased MAPK and Akt signaling. Further, lowering ER stress by 4-phenylbutyrate mitigated the enhanced immune stimulation associated with fatty-acid synthesis blockade. Our findings elucidate the role of fatty-acid synthesis in DC development and function and have implications to the design of DC vaccines for immunotherapy.

Cationic influenza virosomes as an adjuvanted delivery system for CTL induction by DNA vaccination

DNA vaccines have emerged as an attractive approach to induce CTL responses against cancer and infectious agents in recent years. Although CTL induction by DNA vaccination would be a valuable strategy for controlling viral infections, increasing the potency of DNA vaccines is mandatory before DNA vaccines can make it to the clinic. In this study, we developed and characterized a new and safe adjuvanted delivery system for DNA vaccination using cationic influenza virosomes (CIV). CIV were produced by reconstitution of detergent-solubilized influenza virus membranes in the presence of cationic lipids. Plasmid DNA (pDNA) mixed with these virosomes was efficiently transfected into cells of a mouse macrophage cell line (RAW-Blue cells). Moreover, the cells were effectively activated as demonstrated by production of an NFκB/AP-1-inducible reporter enzyme. Following three intradermal immunizations, CIV-delivered epitope-encoding pDNA induced equal numbers of IFNγ- and granzyme B-producing T cells than a 10-fold higher dose of naked pDNA. Virosomes without cationic lipids also improved induction of cellular immunity by pDNA but to a significantly lower extent than CIV. These findings suggest that pDNA–CIV complexes could be an efficacious delivery system suitable for CTL induction by DNA vaccination.

Direct in vivo evidence of CD4+ T cell requirement for CTL response and memory via pMHC-I targeting and CD40L signaling

CD4(+) T cell help contributes critically to DC-induced CD8(+) CTL immunity. However, precisely how these three cell populations interact and how CD4(+) T cell signals are delivered to CD8(+) T cells in vivo have been unclear. In this study, we developed a novel, two-step approach, wherein CD4(+) T cells and antigen-presenting DCs productively engaged one another in vivo in the absence of cognate CD8(+) T cells, after which, we selectively depleted the previously engaged CD4(+) T cells or DCs before allowing interactions of either population alone with naïve CD8(+) T cells. This protocol thus allows us to clearly document the importance of CD4(+) T-licensed DCs and DC-primed CD4(+) T cells in CTL immunity. Here, we provide direct in vivo evidence that primed CD4(+) T cells or licensed DCs can stimulate CTL response and memory, independent of DC-CD4(+) T cell clusters. Our results suggest that primed CD4(+) T cells with acquired pMHC-I from DCs represent crucial "immune intermediates" for rapid induction of CTL responses and for functional memory via CD40L signaling. Importantly, intravital, two-photon microscopy elegantly provide unequivocal in vivo evidence for direct CD4-CD8(+) T cell interactions via pMHC-I engagement. This study corroborates the coexistence of direct and indirect mechanisms of T cell help for a CTL response in noninflammatory situations. These data suggest a new "dynamic model of three-cell interactions" for CTL immunity derived from stimulation by dissociated, licensed DCs, primed CD4(+) T cells, and DC-CD4(+) T cell clusters and may have significant implications for autoimmunity and vaccine design.

The role of splenic CD103+CD8α+cDCs in the induction of CTL responses during respiratory virus infection (168.11)

Abstract We have previously reported that large numbers of virus-specific CD8+ T cells were detected in the spleen following intranasal influenza virus infection. This finding does not seem simply due to the migration of cells from the draining mediastinal lymph nodes (mLN), but rather indicates that spleen can be a site for the induction of primary CTL responses. Although the integrin αEβ7 (CD103)+ migratory DCs have been known as a major DC subset which prime naïve influenza virus-specific CD8+ T cells in the mLN, what kind of DC subset is responsible for the induction of CTL responses in the spleen remains unclear. In this study, we found that increased numbers of CD103+DCs were detectable in the spleen following influenza virus infection, and the majority of CD103+DCs expressed CD8α. Although both CD103+CD11chi DCs (CD103+DCs) and CD103-CD11chi DCs (CD103-DCs) showed activated phenotype evidenced by the expression of co-stimulatory molecules at 9 days post infection, PDL-1 expression pattern was different between these two subsets. Immunohistochemical analysis showed that CD103+DCs localized in the T cell area (also in the B cell area) and purified CD103+DCs produced large amounts of IL-12p40 compared with CD103-DCs. These data suggest that splenic CD103+DCs may play a critical role in the development of CTL responses during influenza virus infection. We are currently investigating how efficiently splenic CD103+DCs act as antigen presenting cells.

Promiscuous Binding of Invariant Chain-Derived CLIP Peptide to Distinct HLA-I Molecules Revealed in Leukemic Cells

Antigen presentation by HLA class I (HLA-I) and HLA class II (HLA-II) complexes is achieved by proteins that are specific for their respective processing pathway. The invariant chain (Ii)-derived peptide CLIP is required for HLA-II-mediated antigen presentation by stabilizing HLA-II molecules before antigen loading through transient and promiscuous binding to different HLA-II peptide grooves. Here, we demonstrate alternative binding of CLIP to surface HLA-I molecules on leukemic cells. In HLA-II-negative AML cells, we found plasma membrane display of the CLIP peptide. Silencing Ii in AML cells resulted in reduced HLA-I cell surface display, which indicated a direct role of CLIP in the HLA-I antigen presentation pathway. In HLA-I-specific peptide eluates from B-LCLs, five Ii-derived peptides were identified, of which two were from the CLIP region. In vitro peptide binding assays strikingly revealed that the eluted CLIP peptide RMATPLLMQALPM efficiently bound to four distinct HLA-I supertypes (-A2, -B7, -A3, -B40). Furthermore, shorter length variants of this CLIP peptide also bound to these four supertypes, although in silico algorithms only predicted binding to HLA-A2 or -B7. Immunization of HLA-A2 transgenic mice with these peptides did not induce CTL responses. Together these data show a remarkable promiscuity of CLIP for binding to a wide variety of HLA-I molecules. The found participation of CLIP in the HLA-I antigen presentation pathway could reflect an aberrant mechanism in leukemic cells, but might also lead to elucidation of novel processing pathways or immune escape mechanisms.

Induction of insert-specific immune response in mice by hamster polyomavirus VP1 derived virus-like particles carrying LCMV GP33 CTL epitope

Hamster polyomavirus (HaPyV) major capsid protein VP1 based chimeric virus-like particles (VLPs) carrying model GP33 CTL epitope derived from Lymphocytic choriomeningitis virus (LCMV) were generated in yeast and examined for their capability to induce CTL response in mice. Chimeric VP1-GP33 VLPs were effectively processed in antigen presenting cells in vitro and in vivo and induced antigen-specific CD8+ T cell proliferation. Mice immunized only once with VP1-GP33 VLPs without adjuvant developed an effective GP33-specific memory T cell response: 70% were fully and 30% partially protected from LCMV infection. Moreover, aggressive growth of tumors expressing GP33 was significantly delayed in these mice in vivo. Therefore, HaPyV VP1-derived VLP harboring CTL epitopes are attractive vaccine candidates for the induction of insert-specific CTL immune response.

ISCOMATRIX Adjuvant Combines Immune Activation with Antigen Delivery to Dendritic Cells In Vivo Leading to Effective Cross-Priming of CD8+ T Cells

Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-γ production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8α(+) DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8α(+) DCs in VDLNs, whereas Langerin(+) DCs and CD8α(-) DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8α(+) DCs in vivo for efficient induction of CTL responses.

Complement as an Endogenous Adjuvant for Dendritic Cell-Mediated Induction of Retrovirus-Specific CTLs

Previous studies have demonstrated the involvement of complement (C) in induction of efficient CTL responses against different viral infections, but the exact role of complement in this process has not been determined. We now show that C opsonization of retroviral particles enhances the ability of dendritic cells (DCs) to induce CTL responses both in vitro and in vivo. DCs exposed to C-opsonized HIV in vitro were able to stimulate CTLs to elicit antiviral activity significantly better than non-opsonized HIV. Furthermore, experiments using the Friend virus (FV) mouse model illustrated that the enhancing role of complement on DC-mediated CTL induction also occurred in vivo. Our results indicate that complement serves as natural adjuvant for DC-induced expansion and differentiation of specific CTLs against retroviruses.

Abstract 1500: Modulation of recombinant vaccinia virus vector immunogenicity

Abstract Recombinant poxviruses expressing tumor associated antigens (TAAs) are evaluated since 20 years as immunogenic vaccine vector in clinical trials. Possible limitation to recombinant viral vector is due to either prior systemic immunity to poxviruses or immunodominance of viral antigens which may reduce the induction of immune response against weaker tumor antigens. To address this issue, we developed a recombinant Vaccinia virus expressing HSV type I protein ICP47. This protein down-regulates MHC class-I antigen presentation by blocking the transporter associated with antigen processing (TAP), which translocates peptides, generated by proteasomal protein degradation, into the endoplasmic reticulum for loading onto MHC class I molecule. Herpes simplex virus (HSV) US12 gene, coding for infected cell protein 47 (ICP47) was introduced into wild type vaccinia virus and into a r.VV expressing MART-1/Melan-A27–35 HLA-A201 endoplasmic reticulum (ER)-targeted epitope. Following infection with non-replicating recombinant virus, effect of ICP47 expression on cell surface MHC-class-I, MHC class-II and co-stimulatory molecules was characterized by antibody staining and FACS analysis. Human T-lymphocytes were stimulated in vitro with autologous CD14+ cells infected with r.VV-US12, r.VV-Mart-US12 or control virus. Responsiveness of specific CD8+ and CD4+ to viral proteins and recombinant epitopes were monitored by MHC-multimer staining and interferon gamma (IFNg) expression analysis. Cells infected with HSV-US12-r.VV, demonstrated a decreased ability of presenting MHC class-I antigens to CD8+ T cells whereas MHC-class-II restricted presentation to CD4+ T cells remained unaffected. Co-expression of ER-Melan-A/Mart-126–35 appeared to partially compensate for the ICP47 related surface MHC class-I molecule down-regulation and preserve a strong capacity to induce CTL response against the TAA derived peptide. Thus, viral vectors expressing ICP47 confirmed a diminished TAP-dependant processing of endogenous class-I restricted epitopes while the immunogenic potential of recombinant epitopes directly targeted to the ER was enhanced. Such reagents could become of high relevance especially in multiple-boost vaccine protocol required in cancer immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1500.

Monophosphoryl lipid A plus IFNgamma maturation of dendritic cells induces antigen-specific CD8+ cytotoxic T cells with high cytolytic potential.

Dendritic cells (DCs) are promising antigen presenting cells for cancer treatment. Previously, we showed that the combination of monophosphoryl lipid A (MPLA) with IFNgamma generates mature DCs that produce IL-12 and polarize CD4(+) T cells towards a Th1 phenotype. Here, we extended these observations by showing that the DCs generated with the clinical grade maturation cocktail of MPLA/IFNgamma induce superior tumour antigen-specific CD8(+) CTL responses compared to the cytokine cocktail matured DCs that are currently used in the clinic. MPLA/IFNgamma DCs can induce CTL responses in healthy individuals as well as in melanoma patients. The CTL induction was mainly dependent on the IL-12 produced by the MPLA/IFNgamma DCs. The high amounts of induced CTLs are functional as they produce IFNgamma and lyse target cells and this cytolytic activity is antigen specific and HLA restricted. Furthermore, the CTLs proved to kill tumour cells expressing endogenous tumour antigen in vitro. Therefore, MPLA/IFNgamma DCs are very promising for the use in future cancer immunotherapy.

Triggering DTH and CTL Activity by fd Filamentous Bacteriophages: Role of CD4+ T Cells in Memory Responses

The ability of fd bacteriophage particles to trigger different arms of the immune system has been previously shown by us with particular emphasis on the ability of phages to raise CTL responses in vitro and in vivo. Here we show that fd virions in the absence of adjuvants are able to evoke a DTH reaction mediated by antigen specific CD8+ T cells. In addition, we analyzed the induction of CTL responses in mice depleted of CD4+ T cells, and we observed that short-term secondary CTL responses were induced in the absence of CD4+ T cells while induction of long-term memory CTLs required the presence of CD4+ T lymphocytes. These results examine the cellular mechanism at the basis of fd efficiency and provide new elements to further validate the use of fd particles for eliciting and monitoring antigen-specific CTLs.

Nonintegrating Lentiviral Vector-Based Vaccine Efficiently Induces Functional and Persistent CD8+ T Cell Responses in Mice

CD8+ T cells are an essential component of an effective host immune response to tumors and viral infections. Genetic immunization is particularly suitable for inducing CTL responses, because the encoded proteins enter the MHC class I processing pathway through either transgene expression or cross-presentation. In order to compare the efficiency and persistence of immune response induced by genetic vaccines, BALB/c mice were immunized either twice intramuscularly with DNA plasmid expressing a codon-optimized HIV-1 gp120 Envelope sequence together with murine GM-CSF sequence or with a single immunization using an integrase defective lentiviral vector (IDLV) expressing the same proteins. Results strongly indicated that the schedule based on IDLV vaccine was more efficient in inducing specific immune response, as evaluated three months after the last immunization by IFNγ ELISPOT in both splenocytes and bone marrow- (BM-) derived cells, chromium release assay in splenocytes, and antibody detection in sera. In addition, IDLV immunization induced high frequency of polyfunctional CD8+ T cells able to simultaneously produce IFNγ, TNFα, and IL2.