A Highly Active Form of XCL1/Lymphotactin Functions as an Effective Adjuvant to Recruit Cross-Presenting Dendritic Cells for Induction of Effector and Memory CD8+ T Cells.

The chemokine receptor XCR1 is known to be selectively expressed by cross-presenting dendritic cells (DCs), while its ligand XCL1/lymphotactin is mainly produced by activated CD8+ T cells and natural killer cells. Recent studies have shown that XCL1-antigen fusion proteins efficiently induce CD8+ T cell responses by preferentially delivering antigens to XCR1+ DCs. However, XCL1 per se was found to be a poor adjuvant for induction of CD8+ T cell responses. XCL1 is unique because of its lack of one of the two disulfide bonds commonly conserved in all other chemokines and thus has an unstable structure with a relatively weak chemokine activity. In the present study, we generated a variant form of murine XCL1 termed mXCL1-V21C/A59C that contained a second disulfide bond to stabilize its chemokine structure. We confirmed that mXCL1-V21C/A59C had much more potent chemotactic and calcium mobilization activities than the wild type XCL1 (mXCL1-WT). Intradermal injection of mXCL1-V21C/A59C, but not that of mXCL1-WT, significantly increased the accumulation of XCR1+CD103+ DCs in the injection site, and most of the accumulated XCR1+CD103+ DCs were found to take up co-injected ovalbumin (OVA). Furthermore, recruited XCR1+CD103+ DCs efficiently migrated to the draining lymph nodes and stayed for a prolonged period of time. Consequently, mXCL1-V21C/A59C strongly induced OVA-specific CD8+ T cells. The combination of OVA and mXCL1-V21C/A59C well protected mice from E.G7-OVA tumor growth in both prophylactic and therapeutic protocols. Finally, memory CTL responses were efficiently induced in mice immunized with OVA and mXCL1-V21C/A59C. Although intradermal injection of OVA and polyinosinic-polycytidylic acid (poly(I:C)) as an adjuvant also induced CD8+ T cell responses to OVA, poly (I:C) poorly recruited XCR1+CD103+ DCs in the injection site and failed to induce significant memory CTL responses to OVA. Collectively, our findings demonstrate that a highly active form of XCL1 is a promising vaccine adjuvant for cross-presenting DCs to induce antigen-specific effector and memory CD8+ T cells.

Porous silicon microparticle potentiates anti-tumor immunity by enhancing cross-presentation and inducing type I interferon response.

Micro- and nanometer-size particles have become popular candidates for cancer vaccine adjuvants. However, the mechanism by which such particles enhance immune responses remains unclear. Here, we report a porous silicon microparticle (PSM)-based cancer vaccine that greatly enhances cross-presentation and activates type I interferon (IFN-I) response in dendritic cells (DCs). PSM-loaded antigen exhibited prolonged early endosome localization and enhanced cross-presentation through both proteasome- and lysosome-dependent pathways. Phagocytosis of PSM by DCs induced IFN-I responses through a TRIF- and MAVS-dependent pathway. DCs primed with PSM-loaded HER2 antigen produced robust CD8 T cell-dependent anti-tumor immunity in mice bearing HER2+ mammary gland tumors. Importantly, this vaccination activated the tumor immune microenvironment with elevated levels of intra-tumor IFN-I and MHCII expression, abundant CD11c+ DC infiltration, and tumor-specific cytotoxic T cell responses. These findings highlight the potential of PSM as an immune adjuvant to potentiate DC-based cancer immunotherapy.

Enhanced endoplasmic reticulum entry of tumor antigen is crucial for cross-presentation induced by dendritic cell-targeted vaccination.

Efficient cross-presentation of protein Ags to CTLs by dendritic cells (DCs) is essential for the success of prophylactic and therapeutic vaccines. In this study, we report a previously underappreciated pathway involving Ag entry into the endoplasmic reticulum (ER) critically needed for T cell cross-priming induced by a DC-targeted vaccine. Directing the clinically relevant, melanoma Ag gp100 to mouse-derived DCs by molecular adjuvant and chaperone Grp170 substantially facilitates Ag access to the ER. Grp170 also strengthens the interaction of internalized protein Ag with molecular components involved in ER-associated protein dislocation and/or degradation, which culminates in cytosolic translocation for proteasome-dependent degradation and processing. Targeted disruption of protein retrotranslocation causes exclusive ER retention of tumor Ag in mouse bone marrow-derived DCs and splenic CD8(+) DCs. This results in the blockade of Ag ubiquitination and processing, which abrogates the priming of Ag-specific CD8(+) T cells in vitro and in vivo. Therefore, the improved ER entry of tumor Ag serves as a molecular basis for the superior cross-presenting capacity of Grp170-based vaccine platform. The ER access and retrotranslocation represents a distinct pathway that operates within DCs for cross-presentation and is required for the activation of Ag-specific CTLs by certain vaccines. These results also reinforce the importance of the ER-associated protein quality control machinery and the mode of the Ag delivery in regulating DC-elicited immune outcomes.

A glucose-6-phosphate isomerase peptide induces T and B cell-dependent chronic arthritis in C57BL/10 mice: arthritis without reactive oxygen species and complement.

Immunization with human glucose-6-phosphate isomerase (hG6PI) protein or with several of its peptides induces arthritis in DBA/1 mice. We investigated G6PI peptide-induced arthritis in C57BL/10 mice and the effect of oxidative burst on disease. To study the arthritogenicity of G6PI peptides and its immune dependency, we used genetically modified and congenic mice on the C57BL/10 background and in vitro T- and B-cell assays. hG6PI(325-339) peptide induced arthritis in C57BL/10 mice. The disease was associated with major histocompatibility complex class II and was dependent on T cells, B cells, and complement C5. Th1 and Th17 cells primed with the hG6PI(325-339) peptide cross-reacted with the murine G6PI protein. The severity of the disease increased in mice carrying a mutation in Ncf1 (Ncf1*/*), which abolishes the NADPH oxidase 2 complex oxidative burst. Ncf1*/* mice developed arthritis also on immunization with the mouse G6PI325-339 peptide and in the absence of C5. The antibody responses to the G6PI protein and peptides were minimal in both Ncf1*/* and wild-type mice. Herein is described G6PI peptide as the first peptide to induce arthritis in C57BL/10 mice. The differences between the wild-type and Ncf1*/* mice suggest that an alternative complement-independent arthritogenic pathway could be operative in the absence of oxidative burst.

Alpha-alumina nanoparticles induce efficient autophagy-dependent cross-presentation and potent antitumour response.

Therapeutic cancer vaccination is an attractive strategy because it induces T cells of the immune system to recognize and kill tumour cells in cancer patients. However, it remains difficult to generate large numbers of T cells that can recognize the antigens on cancer cells using conventional vaccine carrier systems. Here we show that α-Al(2)O(3) nanoparticles can act as an antigen carrier to reduce the amount of antigen required to activate T cells in vitro and in vivo. We found that α-Al(2)O(3) nanoparticles delivered antigens to autophagosomes in dendritic cells, which then presented the antigens to T cells through autophagy. Immunization of mice with α-Al(2)O(3) nanoparticles that are conjugated to either a model tumour antigen or autophagosomes derived from tumour cells resulted in tumour regression. These results suggest that α-Al(2)O(3) nanoparticles may be a promising adjuvant in the development of therapeutic cancer vaccines.

Melanoma-targeted chemo-thermo-immuno (CTI)-therapy using N-propionyl-4-S-cysteaminylphenol-magnetite nanoparticles elicits CTL response via heat shock protein-peptide complex release.

Melanogenesis substrate, N-propionyl-4-S-cysteaminylphenol (NPrCAP) is specifically taken up by melanoma cells and inhibits their growth by producing cytotxic free radicals. By taking advantage of this unique chemical agent, we have established melanoma-targeting intracellular hyperthermia by conjugating NPrCAP with magnetite nanoparticles (NPrCAP/M) upon exposure to an alternating magnetic field (AMF). This treatment causes cytotoxic reaction as well as heat shock responses, leading to elicitation of antitumor immune response, which was proved by tumor rechallenge test and CTL induction. We found the level of heat shock protein 72 (Hsp72) to be increased in the cell lysate and culture supernatant after intracellular hyperthermia. Melanoma-specific CD8(+) T-cell response to dendritic cells loaded with hyperthermia-treated tumor lysate was enhanced when compared with non-treated tumor lysate. When heat shock protein, particularly Hsp72, was immuno-depleted from hyperthermia-treated tumor cell lysate, specific CD8(+) T-cell response was abolished. Thus, it is suggested that antitumor immune response induced by hyperthermia using NPrCAP/M is derived from the release of HSP-peptide complex from degraded tumor cells. Therefore, this chemo-thermo-immuno (CTI)-therapy might be effective not only for primary melanoma but also for distant metastasis because of induction of systemic antimelanoma immune responses.

Anaphylaxis to pine nut: cross-reactivity to Artemisia vulgaris?

The use of pine nuts, the seeds of Pinus pinea, is on the increasing in the modern Mediterranean diet. Little more than 20 cases of allergy to this tree nut have been published, and cross-reactivity with pine pollen, peanut and almond has already been reported. We describe the case of a young boy with several episodes of anaphylaxis after pine nut ingestion. Specific IgE to pine nut and Artemisia vulgaris was demonstrated by skin prick tests and in vitro determination of specific IgE, although no IgE to pine pollen or other nuts was detected. Immunoblotting of Artemisia vulgaris and pine nut revealed two matching diffuse bands, just below 14 kDa and 30 kDa. The ImmunoCAP® inhibition assays showed complete inhibition of pine nut specific IgE after serum incubation with Artemisia vulgaris extract. As far as we know, this is the first reported case of documented cross-reactivity between pine nut and Artemisia vulgaris

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Improving pollen immunotherapy: minor allergens and panallergens

Multiple sensitizations to pollens are common clinical situations in Spain, and alter the efficacy of allergen-specific immunotherapy. We now know that optimization of the diagnosis is required to define the best suited treatment for each patient. All pollen allergens belong to 29 families of proteins ­ the most abundant being the expansins, prophyllins and polcalcins. The ubiquitous nature of proteins such as the prophyllins and polcalcins defines them as panallergens, and explains the cross-reactivity that is erroneously interpreted by clinicians as constituting multi-sensitization. Other families of allergens, such as the calcium transporting proteins (LTPs) are more restricted, but are associated to severe types of allergic disease ­ this being particularly useful to decide upon the indication of immunotherapy. Although recombinant allergens can be produced for in vitro diagnostic purposes, current legislation only allows the use of natural proteins for immunotherapy. However, the same technology can be applied to the study of extracts for vaccines, and it seems that allergen quantification by the manufacturers is a no return trip which clinicians are obliged to follow

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Hyperthermia enhances CTL cross-priming.

Dendritic cells (DCs) loaded with killed allogeneic melanoma cells can cross-prime naive CD8(+) T cells to differentiate into melanoma-specific CTLs in 3-wk cultures. In this study we show that DCs loaded with killed melanoma cells that were heated to 42 degrees C before killing are more efficient in cross-priming of naive CD8(+) T cells than DCs loaded with unheated killed melanoma cells. The enhanced cross-priming was demonstrated by several parameters: 1) induction of naive CD8(+) T cell differentiation in 2-wk cultures, 2) enhanced killing of melanoma peptide-pulsed T2 cells, 3) enhanced killing of HLA-A*0201(+) melanoma cells in a standard 4-h chromium release assay, and 4) enhanced capacity to prevent tumor growth in vitro in a tumor regression assay. Two mechanisms might explain the hyperthermia-induced enhanced cross-priming. First, heat-treated melanoma cells expressed increased levels of 70-kDa heat shock protein (HSP70), and enhanced cross-priming could be reproduced by overexpression of HSP70 in melanoma cells transduced with HSP70 encoding lentiviral vector. Second, hyperthermia resulted in the increased transcription of several tumor Ag-associated Ags, including MAGE-B3, -B4, -A8, and -A10. Thus, heat treatment of tumor cells permits enhanced cross-priming, possibly via up-regulation of both HSPs and tumor Ag expression.

¿Es la sensibilización al polen de la palmera una expresión de la alergia a las profilinas en el área mediterránea? / Specific immunotherapy and respiratory allergic diseases

La palmera datilera (Phoenix dactylifera) es un árbol común en las regiones mediterráneas. En España, las cifras de prevalencia de sensibilización cutánea a este polen oscilan entre el 5,6%, en Elche, y el 29,41%, en Zaragoza, aunque no se han encontrado pacientes monosensibilizados a este polen. Esto hace pensar que la sensibilización a la palmera es la expresión de una reactividad cruzada entre pólenes. Los objetivos del trabajo fueron: 1) estudiar la prevalencia en Cartagena de sensibilización cutánea al polen de la palmera en pacientes con polinosis, uno de cuyos alérgenos es una profilina y correlacionarla con la de Betula alba y Corylus avellana, pólenes que no se encuentran en nuestra zona y que también contienen profilina; 2) estudiar si la distribución de los pacientes, según el número de pólenes a los que estaban sensibilizados, variaba dependiendo de la sensibilización a la palmera; 3) relacionar la sensibilización al polen de la palmera con la sensibilización al melocotón, uno de cuyos alérgenos es una profilina. Material y métodos: Se estudiaron 183 pacientes, 102 varones y 81 mujeres, con una edad media de 28,61 años. En todos se realizaron pruebas cutáneas con pólenes de palmera, abedul, avellano, piel y pulpa de melocotón. Según el número de pólenes a los que estaban sensibilizados se distribuyeron en subgrupos: A (1 a 3), B (4 a 6), C (más de 6). Todos respondieron a un cuestionario sobre si presentaban síntomas al exponerse al melocotón. Resultados: 30 pacientes (16,39%) estaban sensibilizados al polen de la palmera y 29 (96,67%) a las betuláceas; la mayoría se incluía en el subgrupo C, mientras que el mayor porcentaje de los negativos se incluía en el A. El porcentaje de pacientes sensibilizados al melocotón fue mayor en la población sensibilizada a la palmera: 10% frente a 7,84%. Además, el 13,33% de los pacientes sensibilizados a la palmera referían síntomas con la fruta, comparados con el 8,50% de los no sensibilizados. Conclusiones: En nuestra área, la sensibilización al polen de la palmera se asocia a la sensibilización a los pólenes de las betuláceas y al melocotón. Además, los pacientes con pruebas cutáneas positivas a la palmera están sensibilizados a varios pólenes. Esto hace pensar que la sensibilización al polen de la palmera, en el área mediterránea, podría ser la expresión de la sensibilización a un panalérgeno, posiblemente una profilina

Date palm (Phoenix dactylifera) is a common tree in mediterranean area. In Spain, the prevalence of cutaneous sensitization to this pollen range from 5.6% in Elche to 29.41% in Zaragoza, although no patients monosensitized to this pollen have been registered. This suggests that palm pollen sensitization is the expression of a cross-reactivity among pollens. The objectives of this work were: 1) to assess the prevalence in Cartagena of cutaneous sensitization to palm pollen (which contains a profilin allergen) in patients with pollinosis and correlate it with that to Betula alba and Corylus avellana, pollens not found in our area and that contains also profilin; 2) to evaluate if distribution of patients, based on number on pollen sensitizations, varies according to whether or not palm sensitization exists; 3) to relate palm pollen sensitization with peach sensitization, which contain profilin. Material and methods: One hundred an eighty-three patients were studied, 102 men and 81 women, with a mean age of 28.61 years. Skin tests with pollens from palm, birch, hazel tree, peach pulp and peel were performed in all patients. According with the number of pollen to which patients were sensitized, patients were subdivided in: A (1 to 3), B (4 to 6), C (more than 6). All of them filled a questionnaire about symptoms related with peach exposition. Results: 30 patients (16.39%) were sensitized to date palm pollen and 29 (96.67%) to betulaceae; most belonged to group C, where as the higher percentage of negative-test patients were included in group A. The rate of patients sensitized to peach was higher in palm sensitized population: 10% vs. 7.84%. In addition, 13.33% of sensitized patients to palm referred symptoms with the fruit, compared with 8.50% of non sensitized ones. Conclusions: In our area, sensitization to palm pollen is associated with sensitization to betulaceae pollens and peach. Furthermore, patients with positive skin tests to palm pollen are sensitized to various pollens. This suggests that palm pollen sensitization, in the Mediterranean area, could represent the expression of a panallergen sensitization, probably a profilin

Dendritic cell-mediated cross-presentation of antigens derived from colon carcinoma cells exposed to a highly cytotoxic multidrug regimen with gemcitabine, oxaliplatin, 5-fluorouracil, and leucovorin, elicits a powerful human antigen-specific CTL response with antitumor activity in vitro.

Gemcitabine, oxaliplatin, leucovorin, and 5-fluorouracil (GOLF) is a novel multidrug regimen inducing high levels of necrosis and apoptosis in colon carcinoma cells. This regimen is also able to promote a process of Ag remodeling including up-regulation of immunotherapy targets like carcinoembryonic Ag (CEA), thymidylate synthase (TS). We have conducted a preclinical study aimed to investigate whether these drug-induced modifications would also enhance colon cancer cell immunogenicity. Several CTL lines were thus generated by in vitro stimulating human HLA-A(*)02.01(+) PBMCs, from normal donors and colon cancer patients, with autologous dendritic cells cross-primed with cell lysates of colon cancer cells untreated, irradiated, or previously exposed to different drug treatments including the GOLF regimen. Class I HLA-restricted cytolytic activity of these CTL lines was tested against colon cancer cells and CEA and TS gene transfected target cells. These experiments revealed that CTLs sensitized with GOLF-treated cancer cells were much more effective than those sensitized with the untreated colon carcinoma cells or those exposed to the other treatments. CTL lines sensitized against the GOLF-treated colon cancer cells, also expressed a greater percentage of T-lymphocyte precursors able to recognize TS- and CEA-derived peptides. These results suggest that GOLF regimen is a powerful antitumor and immunomodulating regimen that can make the tumor cells a suitable means to induce an Ag-specific CTL response. These results suggest that a rationale combination of GOLF chemotherapy with cytokine-based immunotherapy could generate a chemotherapy-modulated Ag-specific T-lymphocyte response in cancer patients able to destroy the residual disease survived to the cytotoxic drugs.