An optimized protocol for the generation and monitoring of conditional orthotopic lung cancer in the KP mouse model using an adeno-associated virus vector compatible with biosafety level 1.

Background: The inducible Kras/p53 lung adenocarcinoma mouse model, which faithfully recapitulates human disease, is routinely initiated by the intratracheal instillation of a virus-based Cre recombinase delivery system. Handling virus-based delivery systems requires elevated biosafety levels, e.g.

Targeting the MET Receptor Tyrosine Kinase as a Strategy for Radiosensitization in Locoregionally Advanced Head and Neck Squamous Cell Carcinoma.

Radiotherapy (RT) along with surgery is the mainstay of treatment in head and neck squamous cell carcinoma (HNSCC). Radioresistance represents a major source of treatment failure, underlining the urgent necessity to explore and implement effective radiosensitization strategies. The MET receptor widely participates in the acquisition and maintenance of an aggressive phenotype in HNSCC and modulates the DNA damage response following ionizing radiation (IR).

Broad Immune Monitoring and Profiling of T Cell Subsets with Mass Cytometry.

Mass cytometry (cytometry by time-of-flight, CyTOF) is a high-dimensional single-cell analytical technology that allows for highly multiplexed measurements of protein or nucleic acid abundances by bringing together the detection capacity of atomic mass spectroscopy and the sample preparation workflow typical of regular flow cytometry. In 2014 the mass cytometer was adapted for the acquisition of samples from microscopy slides (termed imaging mass cytometry), greatly increasing the applicability of this technology with the inclusion of spatial information. By using antibodies (or other probes) labeled with purified metal isotopes, mass cytometers are currently able to detect more than 50 different parameters at a single-cell level, exceeding the dimensionality of any other flow cytometry methodology currently on the market.

Epicutaneous allergen application preferentially boosts specific T cell responses in sensitized patients.

The effects of epicutaneous allergen administration on systemic immune responses in allergic and non-allergic individuals has not been investigated with defined allergen molecules. We studied the effects of epicutaneous administration of rBet v 1 and rBet v 1 fragments on systemic immune responses in allergic and non-allergic subjects. We conducted a clinical trial in which rBet v 1 and two hypoallergenic rBet v 1 fragments were applied epicutaneously by atopy patch testing (APT) to 15 birch pollen (bp) allergic patients suffering from atopic dermatitis, 5 bp-allergic patients suffering from rhinoconjunctivitis only, 5 patients with respiratory allergy without bp allergy and 5 non-allergic individuals.

High-Dimensional Single-Cell Analysis with Mass Cytometry.

Mass cytometry is an analytical technology that combines the sample preparation workflow typical of flow cytometry and the detection capacity of atomic mass spectroscopy, allowing for highly multiplexed measurements of protein or nucleic acid targets on single cells. In 2014, the mass cytometer was adapted for the acquisition of samples from microscopy slides (termed imaging mass cytometry), greatly increasing the applicability of this technology. By using antibodies (or other probes) labeled with purified metal isotopes, the mass cytometer is able to detect up to 50 different parameters (current practical limit) at the single-cell level, enabling a deep and thorough profiling of individual cells in terms of their cell surface protein phenotype, physiological state, proliferation potential, and many other cell states or features.

Activin-A co-opts IRF4 and AhR signaling to induce human regulatory T cells that restrain asthmatic responses.

Type 1 regulatory T (Tr1) cells play a pivotal role in restraining human T-cell responses toward environmental allergens and protecting against allergic diseases. Still, the precise molecular cues that underlie their transcriptional and functional specification remain elusive. Here, we show that the cytokine activin-A instructs the generation of CD4 T cells that express the Tr1-cell-associated molecules IL-10, inducible T-Cell costimulator (ICOS), lymphocyte activation gene 3 protein (LAG-3), and CD49b, and exert strongly suppressive functions toward allergic responses induced by naive and in vivo-primed human T helper 2 cells.

Frequent occurrence of T cell-mediated late reactions revealed by atopy patch testing with hypoallergenic rBet v 1 fragments.

Background: Late allergic reactions are common in the course of allergen-specific immunotherapy and even occur with allergy vaccines with reduced IgE reactivity.

Objective: We sought to study atopy patch test (APT) reactions and T-cell responses to the recombinant birch pollen allergen Bet v 1 and recombinant hypoallergenic T-cell epitope-containing Bet v 1 fragments in patients with birch pollen allergy with and without atopic dermatitis (AD).

Methods: A clinical study was conducted in 15 patients with birch pollen allergy with AD (group 1), 5 patients with birch pollen allergy without AD (group 2), 5 allergic patients without birch pollen allergy (group 3), and 5 nonallergic subjects (group 4) by performing skin prick tests and APTs with rBet v 1 and hypoallergenic rBet v 1 fragments.

Two functionally distinct subsets of mast cells discriminated By IL-2-independent CD25 activities.

We identified two mast cell subsets characterized by the differential expression of surface CD25 (IL-2Rα) and by different abilities to produce cytokines and to proliferate, both in vitro and in vivo. CD25 can be expressed on the surface of immune cells in the absence of the other chains of the IL-2R, which are indispensable for IL-2 signaling. We show that functional differences between the two mast cell populations were dependent on CD25 itself, which directly modulated proliferation and cytokine responses.

Proteome-wide analysis of HIV-specific naive and memory CD4(+) T cells in unexposed blood donors.

The preexisting HIV-1-specific T cell repertoire must influence both the immunodominance of T cells after infection and immunogenicity of vaccines. We directly compared two methods for measuring the preexisting CD4(+) T cell repertoire in healthy HIV-1-negative volunteers, the HLA-peptide tetramer enrichment and T cell library technique, and show high concordance (r = 0.989).

The who's who of T-cell differentiation: human memory T-cell subsets.

Following antigen encounter and subsequent resolution of the immune response, a single naïve T cell is able to generate multiple subsets of memory T cells with different phenotypic and functional properties and gene expression profiles. Single-cell technologies, first and foremost flow cytometry, have revealed the complex heterogeneity of the memory T-cell compartment and its organization into subsets. However, a consensus has still to be reached, both at the semantic (nomenclature) and phenotypic level, regarding the identification of these subsets.

Holoendemic malaria exposure is associated with altered Epstein-Barr virus-specific CD8(+) T-cell differentiation.

Coinfection with Plasmodium falciparum malaria and Epstein-Barr virus (EBV) is a major risk factor for endemic Burkitt lymphoma (eBL), still one of the most prevalent pediatric cancers in equatorial Africa. Although malaria infection has been associated with immunosuppression, the precise mechanisms that contribute to EBV-associated lymphomagenesis remain unclear. In this study, we used polychromatic flow cytometry to characterize CD8(+) T-cell subsets specific for EBV-derived lytic (BMFL1 and BRLF1) and latent (LMP1, LMP2, and EBNA3C) antigens in individuals with divergent malaria exposure.

T cell responses to known allergen proteins are differently polarized and account for a variable fraction of total response to allergen extracts.

A panel of 133 allergens derived from 28 different sources, including fungi, trees, grasses, weeds, and indoor allergens, was surveyed utilizing prediction of HLA class II-binding peptides and ELISPOT assays with PBMC from allergic donors, resulting in the identification of 257 T cell epitopes. More than 90% of the epitopes were novel, and for 14 allergen sources were the first ever identified to our knowledge. The epitopes identified in the different allergen sources summed up to a variable fraction of the total extract response.

Immunologic and virologic events in early HIV infection predict subsequent rate of progression.

Background: Variability in human immunodeficiency virus (HIV) disease progression cannot be fully predicted by CD4(+) T cell counts or viral load (VL). Because central memory T (T(CM)) cells play a critical role in the pathogenesis of simian immunodeficiency virus disease, we hypothesized that quantifying these cells in early HIV infection could provide prognostic information.

Methods: We measured expression of CD45RO, chemokine (C-C motif) receptor (CCR) 5, CCR7, CD27, and CD28 to enumerate naive and memory subsets in samples from recently infected individuals.

Immunomodulatory effects of the Lutzomyia longipalpis salivary gland protein maxadilan on mouse macrophages.

Infection with Leishmania major is enhanced when the sand fly Lutzomyia longipalpis salivary peptide maxadilan (MAX) is injected along with the parasite. Here we determined the effect that MAX has on the secretion of cytokines and nitric oxide (NO) and on parasite survival in macrophages (MPhis). The cytokines produced by MPhis can enhance a type 1 response, which will increase NO and the killing of intracellular pathogens such as L.