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International audience; Gain-of-function mutations in NLRP3 are responsible for a spectrum of autoinflammatory diseases collectively referred to as “cryopyrin-associated periodic syndromes” (CAPS). Treatment of CAPS patients with IL-1–targeted therapies is effective, confirming a central pathogenic role for IL-1β. However, the specific myeloid cell population(s) exhibiting inflammasome activity and sustained IL-1β production in CAPS remains elusive. Previous reports suggested an important role for mast cells (MCs) in this process. Here, we report that, in mice, gain-of-function mutations in Nlrp3 restricted to neutrophils, and to a lesser extent macrophages/dendritic cells, but not MCs, are sufficient to trigger severe CAPS. Furthermore, in patients with clinically established CAPS, we show that skin-infiltrating neutrophils represent a substantial biological source of IL-1β. Together, our data indicate that neutrophils, rather than MCs, can represent the main cellular drivers of CAPS pathology.

We have assessed the immune-regulatory and adjuvant activities of a synthetic glycolipid, ABX196, a novel analog of the parental compound α-GalCer. As expected, ABX196 demonstrated a measurable and significant adjuvant effect in mice and monkeys with no appreciable toxicity at the doses used to promote immune responses. We performed a phase I/II dose escalation study of ABX196 in healthy volunteers, with the objectives to evaluate its safety profile, as well as its ability to be utilized as an adjuvant in the context of a prophylactic vaccine against hepatitis B. ABX196 was administered at three doses: 0.2, 0.4, and 2.0µg, in fourty-four subjects. In all individuals injected with ABX196, peripheral blood NKT cells displayed hallmarks of activation, and 45% of them had measurable circulating IFN-γ 24 hours after the first administration. More importantly, the addition of ABX196 to the very poorly immunogenic HBs antigen resulted in protective anti-HBs antibody responses in a majority of patients, demonstrating the adjuvant properties of ABX196 in human. Further analysis of the cohort of subjects receiving ABX196 with HBs antigen also indicates that a single injection appears sufficient to provide protection. A limited set of adverse events linked to the systemic delivery of ABX196 and access to the liver, is discussed in the context of formulation and the need to limit transport of ABX196 to secondary lymphoid tissues for maximal efficacy (Eudra-CT 2012-001566-15).

NKT cells are a separate lineage of T lymphocytes that co-express receptors for the T-cell and natural killer (NK) cell lineages. Most NKT cells express a semi-invariant T-cell receptor (TCR), Vα14-Jα18 paired with Vβ8.2, Vβ7 or Vβ2 in mice and Vα24-Jα18/Vβ11 in human [1–5]. These cells are referred to as iNKT cells type I NKT cells, or NKT cells, in contrast to type II NKT cells comprising the remaining NKT cells expressing non-invariant TCR [6]. These cells share phenotypic and functional characteristics of T and NK cells. The phenotype of NKT cells expresses a T-cell receptor αβ (TCRαβ), the CD4 or the CD8 co-receptor or neither of them [double-negative (DN) phenotype], the NK1.1 marker, and some Ly49 receptors [7–10]. Emerging evidence indicates that CD4+ and CD4− iNKT cell subsets are functionally distinct [11–13]. The distribution of iNKT cells has been well studied in mice, and less well in human. Murine iNKT cells represent approximately 0.5 % of the T-cell population in the blood and peripheral lymph nodes (LN), and up to 30 % of T cells in the liver, and this population appears to be times less frequent in humans. However, high and low expressers are found in humans and mice [14–17].

Despite the library of new adjuvants available for use in vaccines, we remain, at present, almost reliant on aluminum-based compounds for clinical use. The increasing use of recombinant subunit vaccines, however, makes the need for improved adjuvant of particular interest. Adjuvants are crucial components of all cancer vaccines whether they are composed of whole cells, proteins or peptides. For the purposes of this article, cellular adjuvant products are defined as adjuvants associated with cellular or T-cell immunity. Several pharmaceutical companies are developing new adjuvants or immune enhancers for the treatment of cancers such as melanoma and non-small-cell lung carcinoma. Several products are being developed and have entered clinical trials either alone or in combination. In this article, we discuss recent adjuvant development and novel cellular adjuvant products for therapeutic cancer vaccines.

NKT cells are a distinct lineage of T lymphocytes that are usually identified by the co-expression of the semi-invariant CD1d-restricted alphabeta TCR and the NK1.1 allelic marker of NK lineage receptors in the C57BL/6 mice and related strains. NKT cells can be subdivided based on CD4/CD8 expression and on tissue of origin. NKT cells express significantly the TCR gene products Valpha24 JalphaQ in humans, the homolog of mouse Valpha14 Jalpha18, paired with Vbeta11, the homolog of mouse Vbeta8.2. NKT cells are most frequent in liver (up to 30% of T cells in mice and approximately 4% of hepatic T cells in human), bone marrow, and thymus and represent a smaller proportion of T cells in other tissues including spleen, lymph nodes, blood, and lung. NKT cells recognize a broad array of glycolipids in the context of CD1d presentation, and many studies have characterized a cascade of functions following in vitro and in vivo stimulation by alpha-GalCer, including production of high levels of immune-regulatory cytokines and bystander activation of several cell types including NK, B, T, and dendritic cells. Both in vitro and in vivo methods have been developed for the study of NKT responses to glycolipid presentation by CD1d. In practice, CD1d-glycolipid-loaded tetramers would most reliably identify these cells. In vitro, splenocytes can be used to monitor cytokine release as this population contains all the cells necessary for sequestering, loading onto CD1d molecules, and presentation of glycolipids to NKT cells. Another system involves the use of NKT cell hybridoma and CD1d coated onto plastic plates to measure responses limited to NKT cells more precisely. In vivo, responses are typically measured by injecting the glycolipid into mice and monitoring plasma cytokine levels or DC maturation in the spleen. This chapter describes methods that can be used to identify NKT cells and to asses in vitro and in vivo their activation and expansion.

NKT cells are a distinct lineage of T lymphocytes that are usually identified by the co-expression of the semi-invariant CD1d-restricted alphabeta TCR and the NK1.1 allelic marker of NK lineage receptors in the C57BL/6 mice and related strains. NKT cells can be subdivided based on CD4/CD8 expression and on tissue of origin. NKT cells express significantly the TCR gene products Valpha24 JalphaQ in humans, the homolog of mouse Valpha14 Jalpha18, paired with Vbeta11, the homolog of mouse Vbeta8.2. NKT cells are most frequent in liver (up to 30% of T cells in mice and approximately 4% of hepatic T cells in human), bone marrow, and thymus and represent a smaller proportion of T cells in other tissues including spleen, lymph nodes, blood, and lung. NKT cells recognize a broad array of glycolipids in the context of CD1d presentation, and many studies have characterized a cascade of functions following in vitro and in vivo stimulation by alpha-GalCer, including production of high levels of immune-regulatory cytokines and bystander activation of several cell types including NK, B, T, and dendritic cells. Both in vitro and in vivo methods have been developed for the study of NKT responses to glycolipid presentation by CD1d. In practice, CD1d-glycolipid-loaded tetramers would most reliably identify these cells. In vitro, splenocytes can be used to monitor cytokine release as this population contains all the cells necessary for sequestering, loading onto CD1d molecules, and presentation of glycolipids to NKT cells. Another system involves the use of NKT cell hybridoma and CD1d coated onto plastic plates to measure responses limited to NKT cells more precisely. In vivo, responses are typically measured by injecting the glycolipid into mice and monitoring plasma cytokine levels or DC maturation in the spleen. This chapter describes methods that can be used to identify NKT cells and to asses in vitro and in vivo their activation and expansion.

BACKGROUND: DC derived-exosomes are nanomeric vesicles harboring functional MHC/peptide complexes capable of promoting T cell immune responses and tumor rejection. Here we report the feasability and safety of the first Phase I clinical trial using autologous exosomes pulsed with MAGE 3 peptides for the immunization of stage III/IV melanoma patients. Secondary endpoints were the monitoring of T cell responses and the clinical outcome. PATIENTS AND METHODS: Exosomes were purified from day 7 autologous monocyte derived-DC cultures. Fifteen patients fullfilling the inclusion criteria (stage IIIB and IV, HLA-A1+, or -B35+ and HLA-DPO4+ leukocyte phenotype, tumor expressing MAGE3 antigen) were enrolled from 2000 to 2002 and received four exosome vaccinations. Two dose levels of either MHC class II molecules (0.13 versus 0.40 x 1014 molecules) or peptides (10 versus 100 mug/ml) were tested. Evaluations were performed before and 2 weeks after immunization. A continuation treatment was performed in 4 cases of non progression. RESULTS: The GMP process allowed to harvest about 5 x 1014 exosomal MHC class II molecules allowing inclusion of all 15 patients. There was no grade II toxicity and the maximal tolerated dose was not achieved. One patient exhibited a partial response according to the RECIST criteria. This HLA-B35+/A2+ patient vaccinated with A1/B35 defined CTL epitopes developed halo of depigmentation around naevi, a MART1-specific HLA-A2 restricted T cell response in the tumor bed associated with progressive loss of HLA-A2 and HLA-BC molecules on tumor cells during therapy with exosomes. In addition, one minor, two stable and one mixed responses were observed in skin and lymph node sites. MAGE3 specific CD4+ and CD8+ T cell responses could not be detected in peripheral blood. CONCLUSION: The first exosome Phase I trial highlighted the feasibility of large scale exosome production and the safety of exosome administration.