Characterization of monocyte-derived IFNalpha-generated dendritic cells.

The antitumor effects of IFNalpha is mainly mediated by the activation of cytotoxic T lymphocytes (CTLs), the activation of natural killer (NK) cells, and the generation of highly potent antigen-presenting dendritic cells (IFN-DCs). Recently, we demonstrated that these cells partially express the NK cell marker CD56 and reveal a direct cytotoxic immunity towards tumor cells. The aim of the present study was to explore these cells in more detail with respect to their phenotypical and functional characteristics. Flowcytometric analyses revealed that a 5-day incubation time of CD14+ monocytes with IFNalpha results in a steady increase of CD56 surface expression of these cells from 25% (+/-2%) on day 1 up to 68% (+/-11%) on day 5. Interestingly, additional culturing of negatively selected CD56- IFN-DCs also resulted in a partial CD56 surface expression. By comparing both cell types in more detail we found a significant decrease of CD14 expression on CD56+ IFN-DCs (66+/-6%) compared to CD56- IFN-DCs (76+/-6%). On the basis of functional tests, CD56+ IFN-DCs revealed a slightly increased phagocytosis capacity compared to CD56- IFN-DCs as only 82% of CD56- IFN-DCs showed a positive intracytoplasmatic signal after 60 minutes coculturing with FITC-labeled albumin, whereas 91% of CD56+ IFN-DCs were positive. Moreover, CD56+ IFN-DCs revealed a stronger T cell stimulation capacity compared to CD56- IFN-DCs. These results together with our previously described data suggest that CD56+ IFN-DCs and CD56- IFN-DCs may represent one identical cell population with different maturation status rather than two separate cell entities. Because of their high stimulating capacity and their direct cytolytic effects these cells represent a new promising tool for cellular anticancer therapy.     

IFN-alpha skews monocytes into CD56+-expressing dendritic cells with potent functional activities in vitro and in vivo

The antitumor effect of IFN-alpha is mediated by the activation of CTLs, NK cells, and the generation of highly potent Ag-presenting dendritic cells (IFN-DCs). In this study, we show that IFN-DCs generated in vitro from monocytes express CD56 on their surface, a marker which has been thought to be specific for NK cells. FACS analyses of CD56(+) and CD56(-) IFN-DCs showed a nearly identical pattern for most of the classical DC markers. Importantly, however, only CD56(+) IFN-DCs exhibited cytolytic activity up to 24% that could almost completely be blocked (-81%) after coincubation with anti-TRAIL. Intracytoplasmatic cytokine staining revealed that the majority of IFN-DCs independently of their CD56 expression were IFN-gamma positive as well. In contrast, CD56(+) IFN-DCs showed stronger capacity in stimulating allogenic T cells compared with CD56(-) IFN-DC. Based on these results, five patients with metastasized medullary thyroid carcinoma were treated for the first time with monocyte-derived tumor Ag-pulsed IFN-DCs. After a long term follow-up (in mean 37 mo) all patients are alive. Immunohistochemical analyses of delayed-type hypersensitivity skin reaction showed a strong infiltration with CD8(+) cells. In two patients no substantial change in tumor morphology was detected. Importantly, by analyzing PBMCs, these patients also showed an increase of Ag-specific IFN-gamma-secreting T cells. In summary, we here describe for the first time that cytotoxic activity of IFN-DCs is mainly mediated by an IFN-DC subset showing partial phenotypic and functional characteristics of NK cells. These cells represent another mechanism of the antitumor effect induced by IFN-alpha.     

Administration of a CD25-directed immunotoxin, LMB-2, to patients with metastatic melanoma induces a selective partial reduction in regulatory T cells in vivo

CD25+ CD4+ T regulatory (Treg) cells regulate peripheral self tolerance and possess the ability to suppress antitumor responses, which may in part explain the poor clinical response of cancer patients undergoing active immunization protocols. We have previously shown that in vitro incubation of human PBMC with LMB-2, a CD25-directed immunotoxin, significantly reduced CD25+ FOXP3+ CD4+ Treg cells without impairing the function of the remaining lymphocytes. In the current study, eight patients with metastatic melanoma were treated with LMB-2 followed by MART-1 and gp100-specific peptide vaccination. LMB-2 administration resulted in a preferential, transient reduction in mean circulating CD25+ CD4+ T cell number, from 83 +/- 16 cells/microl to a nadir of 17 +/- 5 cells/microl, a 79.1% reduction. FOXP3 analysis revealed a less robust depletion with mean FOXP3+ CD4+ Treg cell number decreasing from 74 +/- 15 cells/microl to 36 +/- 8 cells/microl, a 51.4% reduction. FOXP3+ CD4+ Treg cells that survived LMB-2-mediated cytotoxicity expressed little or no CD25. Similar to the peripheral blood, immunohistochemical analysis showed a 68.9% mean reduction in FOXP3+ CD4+ Treg cell frequency in evaluable lesions. Despite inducing a reduction in Treg cell numbers in vivo, LMB-2 therapy did not augment the immune response to cancer vaccination and no patient experienced an objective response or autoimmunity. These data demonstrate the capacity of a CD25-directed immunotoxin to selectively mediate a transient partial reduction in circulating and tumor-infiltrating Treg cells in vivo, and suggest that more comprehensive Treg cell elimination may be required to bolster antitumor responses in patients with metastatic melanoma.     

Role of regulatory T cells for the treatment of type 1 diabetes mellitus.

Beta-cell specific autoreactive T cells can be found in patients with type I diabetes (T1D) and in healthy controls. They are usually controlled by a network of regulatory mechanisms including CD4+CD25+Foxp3+ regulatory T cells (Tregs). It was suspected that defects in Treg number and activity are causally related to the development of T1D. Although there are hints that this concept might be true, it is neither proven in animal models nor in patients with T1D. However, increasing the number of Tregs by adoptive transfer can be used to prevent and treat even established T1D. It was demonstrated that Tregs recognizing beta-cell antigens are far more efficient in treating the disease than polyspecific Tregs. The use of beta-cell specific Tregs is also leading to a tissue specific immunotolerance without perturbing the general immunocompetence. Two sources for beta-cell specific Tregs are currently employed: First natural Tregs specific for beta-cells are expanded IN VITRO and reinfused into diabetic animals. Second na?ve or activated T cells specific for beta-cell antigens are IN VITRO converted to Tregs by genetic manipulation or by specific cytokine combinations. Both approaches were successful in treating even established diabetes in animal models. Before such therapies can be used in patients safety measures regarding the fate and the effects of the transferred Tregs have to be studied. Besides this ethical considerations are important in regard to what risks we should take to treat a disease in young patients which can otherwise be treated medically. In the meantime the concept of Tregs for therapy of T1D is supported by successful clinical attempts to induce these cells IN VIVO by administration of monoclonal antibodies against CD3. If subsequent studies show that Tregs represent a safe and efficient source for therapy, they could become an important weapon in the fight against immune mediated pathology.     

Functional analysis of tumor-specific Th cell responses detected in melanoma patients after dendritic cell-based immunotherapy

Recently, we have demonstrated that tumor-specific CD4+ Th cell responses can be rapidly induced in advanced melanoma patients by vaccination with peptide-loaded monocyte-derived dendritic cells. Most patients showed a T cell reactivity against a melanoma Ag 3 (MAGE-3) peptide (MAGE-3(243-258)), which has been previously found to be presented by HLA-DP4 molecules. To analyze the functional and specificity profile of this in vivo T cell response in detail, peptide-specific CD4+ T cell clones were established from postvaccination blood samples of two HLA-DP4 patients. These T cell clones recognized not only peptide-loaded stimulator cells but also dendritic cells loaded with a recombinant MAGE-3 protein, demonstrating that these T cells were directed against a naturally processed MAGE-3 epitope. The isolated CD4+ Th cells showed a typical Th1 cytokine profile upon stimulation. From the first patient several CD4+ T cell clones recognizing the antigenic peptide used for vaccination in the context of HLA-DP4 were obtained, whereas we have isolated from the second patient CD4+ T cell clones which were restricted by HLA-DQB1*0604. Analyzing a panel of truncated peptides revealed that the CD4+ T cell clones recognized different core epitopes within the original peptide used for vaccination. Importantly, a DP4-restricted T cell clone was stimulated by dendritic cells loaded with apoptotic or necrotic tumor cells and even directly recognized HLA class II- and MAGE-3-expressing tumor cells. Moreover, these T cells exhibited cytolytic activity involving Fas-Fas ligand interactions. These findings support that vaccination-induced CD4+ Th cells might play an important functional role in antitumor immunity.     

Monocyte-derived dendritic cells generated after a short-term culture with IFN-alpha and granulocyte-macrophage colony-stimulating factor stimulate a potent Epstein-Barr virus-specific CD8+ T cell response

Cellular immune responses are crucial for the control of EBV-associated lymphoproliferative diseases. To induce an anti-EBV cell-mediated immunity, we have used dendritic cells (DCs) generated by a 3-day culture of human CD14(+) monocytes in the presence of GM-CSF and type I IFN (IFN-DCs) and pulsed with peptides corresponding to CTL EBV epitopes. The functional activity of IFN-DCs was compared with that of APCs differentiated by culturing monocytes for 3 days with GM-CSF and IL-4 and indicated as IL-4-DCs. Stimulation of PBLs from EBV-seropositive donors with EBV peptide-pulsed autologous IFN-DCs resulted in a stronger expansion of specific T lymphocytes producing IFN-gamma with respect to stimulation with peptide-loaded IL-4-DCs, as assessed by ELISPOT assays. When purified CD8(+) T cells were cocultured with EBV peptide-pulsed IFN-DCs or IL-4-DCs, significantly higher levels of specific cytotoxic activity were observed in CD8(+) T cell cultures stimulated with IFN-DCs. Injection of peptide-pulsed IFN-DCs into SCID mice transplanted with autologous PBLs led to the recovery of a significantly greater number of EBV-specific human CD8(+) T cells from the spleen and the peritoneal cavity with respect to that recovered from mice injected with peptide-pulsed IL-4-DCs. Moreover, a significant delay in lymphoma development was observed when peptide-pulsed IFN-DCs were injected into SCID mice reconstituted with PBMCs endowed with a high capability of lymphoma induction, whereas injection of unpulsed IFN-DCs was ineffective. Our results indicate that IFN-DCs efficiently promote in vitro and in vivo the expansion of CD8(+) T lymphocytes acting as cytotoxic effectors against EBV-transformed cells.     

Role of cross-talk between IFN-alpha-induced monocyte-derived dendritic cells and NK cells in priming CD8+ T cell responses against human tumor antigens

In the present study we evaluated the role of IFN-alpha in the generation of dendritic cells (IFN-DCs) with priming activity on CD8(+) T lymphocytes directed against human tumor Ags. A 3-day treatment of monocytes, obtained as adherent PBMCs from HLA-A*0201(+) healthy donors, with IFN-alpha and GM-CSF led to the differentiation of DCs displaying a semimature phenotype, but promptly inducing CD8(+) T cell responses after one in vitro sensitization with peptides derived from melanoma (gp100(209-217) and MART-1/Melan-A(27-35)) and adenocarcinoma (CEA(605-613)) Ags. However, these features were lost when IFN-DCs were generated from immunosorted CD14(+) monocytes. The ability of adherent PBMCs to differentiate into IFN-DCs expressing higher levels of costimulatory molecules and exerting efficient T cell priming capacity was associated with the presence of contaminating NK cells, which underwent phenotypic and functional activation upon IFN-alpha treatment. NK cell boost appeared to be mediated by both direct and indirect (i.e., mediated by IFN-DCs) mechanisms. Experiments performed to prove the role of contaminating NK cells in DC differentiation showed that IFN-DCs generated in the absence of NK were phenotypically less mature and could not efficiently prime antitumor CD8(+) lymphocytes. Reciprocally, IFN-DCs raised from immunosorted CD14(+) monocytes regained their T cell priming activity when NK cells were added to the culture before IFN-alpha and GM-CSF treatment. Together, our data suggest that the ability of IFN-DCs to efficiently prime anti-tumor CD8(+) T lymphocytes relied mostly on the positive cross-talk occurring between DCs and NK cells upon stimulation with IFN-alpha.     

Everolimus is an active agent in medullary thyroid cancer: a clinical and in vitro study

Everolimus, an mTOR inhibitor, which has been demonstrated to induce anti-tumour effects in different types of neuroendocrine tumours, has never been evaluated in patients with medullary thyroid cancer (MTC). The aim of this study was to evaluate the in vitro and in vivo effects of everolimus in combination with octreotide in MTC. Two patients with progressive metastatic MTC and high calcitonin levels were treated with everolimus 5-10 mg/day. Both patients were under treatment with octreotide LAR at the study entry. An in vitro study was also performed to assess everolimus effects on MTC cell lines (TT and MZ-CRC-1 cells). A tumour response was observed in both patients. Serum calcitonin decreased by 86% in patient 1 and by 42% in patient 2. In TT and MZ-CRC-1 cells, everolimus induced a significant dose-dependent inhibition in cell proliferation. This effect seems to be related to a cell cycle arrest in G(0) /G(1) phase in both cell lines and to the induction of cellular senescence in TT cells. Everolimus in combination with octreotide may be active as anti-tumour therapy in patients with progressive metastatic MTC, suggesting to further evaluate this agent in MTC patients in a large prospective study.     

Exposure to Leishmania major modulates the proportion of CD4+ T cells without affecting cellular immune responses

The immune responses of individuals exposed to Leishmania major were evaluated and compared with those of non-exposed volunteers. Forty-one patients with active lesion(s), 43 healed individuals, 15 vaccinees 1 month or 1 year post vaccination, and 15 non-exposed volunteers were studied. Leishmanin skin test (LST) response, proliferative response of lymphocyte (PRL) to L. major antigen, IFN-gamma and IL-4 production, and percentage of L. major-specific CD4+, CD8+ and CD16+/CD56+ cells in peripheral blood mononuclear cells were assessed. Data showed positive LST (>5 mm) in 92% of patients, 98% of healed, and 80% or 43% of vaccinees 1 month and 1 year post vaccination, respectively. Positive PRL (SI>2.5) was displayed in 90%, 84%, 46% and 7% of patients, healed, vaccinated (post 1 year) and non-exposed donors, respectively. The mean +/-S.E. of IFN-gamma was 924 +/- 149, 1,278 +/- 185, 470 +/- 282 or 258 +/- 82 pg/ml in patients, healed cases and vaccinees after 1 month or 1 year, respectively. Positive IFN-gamma responders (>300 pg/ml) were shown in 72% of patients, 81% of healed cases, 31% or 39% of vaccinees and 0% of non-exposed donors. A reduced percentage of CD4+ T-cells and an increased percentage of NK cells were found in exposed individuals compared to non-exposed donors. The data indicated that exposure to L. major modulates the proportion of CD4+ T cells and increases NK cells percentage. However, the cellular immune responses including induction of LST, and IFN-gamma production are increased in exposed individuals.     

IFN-alpha skews monocyte differentiation into Toll-like receptor 7-expressing dendritic cells with potent functional activities

IFN-alpha is an important cytokine for the generation of a protective T cell-mediated immune response to viruses. In this study, we asked whether IFN-alpha can regulate the functional properties of dendritic cells (DCs). We show that monocytes cultured in the presence of GM-CSF and IFN-alpha can differentiate into DCs (IFN-alpha-derived DCs (IFN-DCs)). When compared with DCs generated in the presence of GM-CSF and IL-4 (IL-4-derived DCs), IFN-DCs exhibited a typical DC morphology and expressed, in addition to DC markers CD1a and blood DC Ag 4, a similar level of costimulatory and class II MHC molecules, but a significantly higher level of MHC class I molecules. After maturation with CD40 ligand, IFN-DCs up-regulated costimulatory, class I and II MHC molecules and expressed mature DC markers such as CD83 and DC-lysosome-associated membrane protein. IFN-DCs were endowed with potent functional activities. IFN-DCs secreted large amounts of the inflammatory cytokines IL-6, IL-10, TNF-alpha, IL-1beta, and IL-18, and promoted a Th1 response that was independent of IL-12p70 and IL-18, but substantially inhibited by IFN-alpha neutralization. Furthermore, immature IFN-DCs induced a potent autologous Ag-specific immune response, as evaluated by IFN-gamma secretion and expansion of CD8(+) T cells specific for CMV. Also, IFN-DCs expressed a large number of Toll-like receptors (TLRs), including acquisition of TLR7, which is classically found on the natural type I IFN-producing plasmacytoid DCs. Like plasmacytoid DCs, IFN-DCs could secrete IFN-alpha following viral stimulation or TLR7-specific stimulation. Taken together, these results illustrate the critical role of IFN-alpha at the early steps of immune response to pathogens or in autoimmune diseases.     

Lymphocyte subset analyses in healthy adults vaccinated with yellow fever 17DD virus

In this study the kinetics of humoral and cellular immune responses in first-time vaccinees and re-vaccinees with the yellow fever 17DD vaccine virus was analyzed. Flow cytometric analyses were used to determine percentual values of T and B cells in parallel to the yellow fever neutralizing antibody production. All lymphocyte subsets analyzed were augmented around the 30th post vaccination day, both for first-time vaccinees and re-vaccinees. CD3+ T cells increased from 30.8% (SE +/- 4%) to 61.15% (SE +/- 4.2%), CD4+ T cells from 22.4% (SE +/- 3.6%) to 39.17% (SE +/- 2%) with 43% of these cells corresponding to CD4+CD45RO+ T cells, CD8+ T cells from 15.2% (SE +/- 2.9%) to 27% (SE +/- 3%) with 70% corresponding to CD8+CD45RO+ T cells in first-time vaccinees. In re-vaccinees, the CD3+ T cells increased from 50.7% (SE +/- 3%) to 80% (SE +/- 2.3%), CD4+ T cells from 24.9% (SE +/- 1.4%) to 40% (SE +/- 3%) presenting a percentage of 95% CD4+CD45RO+ T cells, CD8+ T cells from 19.7% (SE +/- 1.8%) to 25% (SE +/- 2%). Among CD8+CD38+ T cells there could be observed an increase from 15 to 41.6% in first-time vaccinees and 20.7 to 62.6% in re-vaccinees. Regarding neutralizing antibodies, the re-vaccinees presented high titers even before re-vaccination. The levels of neutralizing antibodies of first-time vaccinees were similar to those presented by re-vaccinees at day 30 after vaccination, indicating the success of primary vaccination. Our data provide a basis for further studies on immunological behavior of the YF 17DD vaccine.     

In vitro and in vivo induction of a Th cell response toward peptides of the melanoma-associated glycoprotein 100 protein selected by the TEPITOPE program

The melanoma-associated Ag glycoprotein 100 was analyzed by the T cell epitope prediction software TEPITOPE. Seven HLA-DR promiscuous peptides predicted with a stringent threshold were used to load dendritic cells (DC), and induction of a proliferative response was monitored. PBMC of all nine donors including two patients with malignant melanoma responded to at least one of the peptides. The proliferative response was defined as a Th response by the selective expansion of CD4(+) cells, up-regulation of CD25 and CD40L, and IL-2 and IFN-gamma expression. Peptide-loaded DC also initiated a T helper response in vivo (i.e., tumor growth in the SCID mouse was significantly retarded by the transfer of PBMC together with peptide-loaded DC). Because the use of the TEPITOPE program allows for a prediction of T cell epitopes; because the predicted peptides can be rapidly confirmed by inducing a Th response in the individual patient; and because application of peptide-loaded DC suffices for the in vivo activation of helper cells, vaccination with MHC class II-binding peptides of tumor-associated Ags becomes a feasible and likely powerful tool in the immunotherapy of cancer.     

Calcitonin-specific antitumor immunity in medullary thyroid carcinoma following dendritic cell vaccination

In this study, we investigated the immune response following immunotherapy with calcitonin-pulsed dendritic cells (DC) in 7 patients with metastasized medullary thyroid carcinoma. After immunization with 1-5 x 10(6) autologous DC, significant calcitonin-specific T cell proliferation was detectable in 3 patients. Measurement of cytokine release from T lymphocytes demonstrated high post-treatment interferon-gamma (IFN-gamma) secretion after stimulation with calcitonin in 5 patients, one of whom experienced significant tumor regression. In contrast, antigen-specific interleukin-4 (IL-4) production was only slightly increased in 4 patients. All 7 patients developed a strong delayed-type hypersensitivity (DTH) skin reaction, which was confirmed to be mediated by infiltrating CD4+ T-helper cells and CD8+ cytotoxic T cells in all 3 patients who underwent skin biopsy. This is the first study to show that a polypeptide hormone can be used to develop a DC vaccination strategy for the immunotherapy of highly malignant endocrine cancers.     

A long peptide from MELOE-1 contains multiple HLA class II T cell epitopes in addition to the HLA-A*0201 epitope: an attractive candidate for melanoma vaccination

CD4⁺ T cells contribute importantly to the antitumor T cell response, and thus, long peptides comprising CD4 and CD8 epitopes may be efficient cancer vaccines. We have previously identified an overexpressed antigen in melanoma, MELOE-1, presenting a CD8⁺ T cell epitope, MELOE-1_36–44, in the HLA-A*0201 context. A T cell repertoire against this epitope is present in HLA-A*0201+ healthy subjects and melanoma patients and the adjuvant injection of TIL containing MELOE-1 specific CD8⁺ T cells to melanoma patients was shown to be beneficial. In this study, we looked for CD4⁺ T cell epitopes in the vicinity of the HLA-A*0201 epitope. Stimulation of PBMC from healthy subjects with MELOE-1_26–46 revealed CD4 responses in multiple HLA contexts and by cloning responsive CD4⁺ T cells, we identified one HLA-DRβ1*1101-restricted and one HLA-DQβ1*0603-restricted epitope. We showed that the two epitopes could be efficiently presented to CD4⁺ T cells by MELOE-1-loaded dendritic cells but not by MELOE-1+ melanoma cell-lines. Finally, we showed that the long peptide MELOE-1_22–46, containing the two optimal class II epitopes and the HLA-A*0201 epitope, was efficiently processed by DC to stimulate CD4⁺ and CD8⁺ T cell responses in vitro, making it a potential candidate for melanoma vaccination.     

DNA fusion vaccines induce targeted epitope-specific CTLs against minor histocompatibility antigens from a normal or tolerized repertoire

We have designed DNA fusion vaccines able to induce high levels of epitope-specific CD8(+) T cells, using linked CD4(+) T cell help. Such vaccines can activate effective immunity against tumor Ags. To model performance against minor histocompatibility (H) Ags important in allogeneic hemopoietic stem cell transplantation, responses against the H2D(b)-restricted Uty and Smcy male HY epitopes have been investigated. Vaccination of females induced high levels of tetramer-specific, IFN-gamma-producing CD8(+) T cells against each epitope. Vaccines incorporating a single epitope primed effector CTL able to kill male splenocytes in vitro and in vivo, and HY(Db)Uty-specific vaccination accelerated rejection of syngeneic male skin grafts. Priming against either epitope established long-term memory, expandable by injection of male cells. Expanded CD8(+) T cells remained specific for the priming HY epitope, with responses to the second suppressed. To investigate vaccine performance in a tolerized repertoire, male mice were vaccinated with the fusion constructs. Strikingly, this also generated epitope-specific IFN-gamma-producing CD8(+) T cells with cytotoxic function. However, numbers and avidity were lower than in vaccinated females, and vaccinated males failed to reject CFSE-labeled male splenocytes in vivo. Nevertheless, these findings indicate that DNA fusion vaccines can mobilize CD8(+) T cells against endogenous minor H Ags, even from a profoundly tolerized repertoire. In the transplantation setting, vaccination of donors could prime and expand specific T cells for in vivo transfer. For patients, vaccination could activate a potentially less tolerized repertoire against similar Ags that may be overexpressed by tumor cells, for focused immune attack.     

Synergistic antitumour activity of RAF265 and ZSTK474 on human TT medullary thyroid cancer cells

Medullary thyroid cancer (MTC) is an aggressive malignancy responsible for up to 14% of all thyroid cancer‐related deaths. It is characterized by point mutations in the rearranged during transfection (RET) proto‐oncogene. The activated RET kinase is known to signal via extracellular signal regulated kinase (ERK) and phosphoinositide 3‐kinase (PI3K), leading to enhanced proliferation and resistance to apoptosis. In the present work, we have investigated the effect of two serine/threonine‐protein kinase B‐Raf (BRAF) inhibitors (RAF265 and SB590885), and a PI3K inhibitor (ZSTK474), on RET‐mediated signalling and proliferation in a MTC cell line (TT cells) harbouring the RETC634W activating mutation. The effects of the inhibitors on VEGFR2, PI3K/Akt and mitogen‐activated protein kinases signalling pathways, cell cycle, apoptosis and calcitonin production were also investigated. Only the RAF265+ ZSTK474 combination synergistically reduced the viability of treated cells. We observed a strong decrease in phosphorylated VEGFR2 for RAF265+ ZSTK474 and a signal reduction in activated Akt for ZSTK474. The activated ERK signal also decreased after RAF265 and RAF265+ ZSTK474 treatments. Alone and in combination with ZSTK474, RAF265 induced a sustained increase in necrosis. Only RAF265, alone and combined with ZSTK474, prompted a significant drop in calcitonin production. Combination therapy using RAF265 and ZSTK47 proved effective in MTC, demonstrating a cytotoxic effect. As the two inhibitors have been successfully tested individually in clinical trials on other human cancers, our preclinical data support the feasibility of their combined use in aggressive MTC.     

Pitfalls of vaccinations with WT1-, Proteinase3- and MUC1-derived peptides in combination with MontanideISA51 and CpG7909

T cells with specificity for antigens derived from Wilms Tumor gene (WT1), Proteinase3 (Pr3), and mucin1 (MUC1) have been demonstrated to lyse acute myeloid leukemia (AML) blasts and multiple-myeloma (MM) cells, and strategies to enhance or induce such tumor-specific T cells by vaccination are currently being explored in multiple clinical trials. To test safety and immunogenicity of a vaccine composed of WT1-, Pr3-, and MUC1-derived Class I-restricted peptides and the pan HLA-DR T helper cell epitope (PADRE) or MUC1-helper epitopes in combination with CpG7909 and MontanideISA51, four patients with AML and five with MM were repetitively vaccinated. No clinical responses were observed. Neither pre-existing nor naive WT1-/Pr3-/MUC1-specific CD8+ T cells expanded in vivo by vaccination. In contrast, a significant decline in vaccine-specific CD8+ T cells was observed. An increase in PADRE-specific CD4+ T helper cells was observed after vaccination but these appeared unable to produce IL2, and CD4+ T cells with a regulatory phenotype increased. Taken into considerations that multiple clinical trials with identical antigens but different adjuvants induced vaccine-specific T cell responses, our data caution that a vaccination with leukemia-associated antigens can be detrimental when combined with MontanideISA51 and CpG7909. Reflecting the time-consuming efforts of clinical trials and the fact that 1/3 of ongoing peptide vaccination trails use CpG and/or Montanide, our data need to be taken into consideration.     

Targeting of Non-Dominant Antigens as a Vaccine Strategy to Broaden T-Cell Responses during Chronic Viral Infection

In this study, we compared adenoviral vaccine vectors with the capacity to induce equally potent immune responses against non-dominant and immunodominant epitopes of murine lymphocytic choriomeningitis virus (LCMV). Our results demonstrate that vaccination targeting non-dominant epitopes facilitates potent virus-induced T-cell responses against immunodominant epitopes during subsequent challenge with highly invasive virus. In contrast, when an immunodominant epitope was included in the vaccine, the T-cell response associated with viral challenge remained focussed on that epitope. Early after challenge with live virus, the CD8+ T cells specific for vaccine-encoded epitopes, displayed a phenotype typically associated with prolonged/persistent antigenic stimulation marked by high levels of KLRG-1, as compared to T cells reacting to epitopes not included in the vaccine. Notably, this association was lost over time in T cells specific for the dominant T cell epitopes, and these cells were fully capable of expanding in response to a new viral challenge. Overall, our data suggests a potential for broadening of the antiviral CD8+ T-cell response by selecting non-dominant antigens to be targeted by vaccination. In addition, our findings suggest that prior adenoviral vaccination is not likely to negatively impact the long-term and protective immune response induced and maintained by a vaccine-attenuated chronic viral infection.     

Induction of potent CD8+ T-cell responses by novel biodegradable nanoparticles carrying human immunodeficiency virus type 1 gp120

The mainstream of recent anti-AIDS vaccines is a prime/boost approach with multiple doses of the target DNA of human immunodeficiency virus type 1 (HIV-1) and recombinant viral vectors. In this study, we have attempted to construct an efficient protein-based vaccine using biodegradable poly(gamma-glutamic acid) (gamma-PGA) nanoparticles (NPs), which are capable of inducing potent cellular immunity. A significant expansion of CD8+ T cells specific to the major histocompatibility complex class I-restricted gp120 epitope was observed in mice intranasally immunized once with gp120-carrying NPs but not with gp120 alone or gp120 together with the B-subunit of cholera toxin. Both the gp120-encapsulating and -immobilizing forms of NPs could induce antigen-specific spleen CD8+ T cells having a functional profile of cytotoxic T lymphocytes. Long-lived memory CD8+ T cells could also be elicited. Although a substantial decay in the effector memory T cells was observed over time in the immunized mice, the central memory T cells remained relatively constant from day 30 to day 238 after immunization. Furthermore, the memory CD8+ T cells rapidly expanded with boosting with the same immunogen. In addition, gamma-PGA NPs were found to be a much stronger inducer of antigen-specific CD8+ T-cell responses than nonbiodegradable polystyrene NPs. Thus, gamma-PGA NPs carrying various HIV-1 antigens may have great potential as a novel priming and/or boosting tool in current vaccination regimens for the induction of cellular immune responses.