Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8(+) T cells with detection by ELISPOT and HLA-multimer staining.

Ex vivo ELISPOT and multimer staining are well-established tests for the assessment of antigen-specific T cells. Many laboratories are now using a period of in vitro stimulation (IVS) to enhance detection. Here, we report the findings of a multi-centre panel organised by the Association for Cancer Immunotherapy Immunoguiding Program to investigate the impact of IVS protocols on the detection of antigen-specific T cells of varying ex vivo frequency. Five centres performed ELISPOT and multimer staining on centrally prepared PBMCs from 3 donors, both ex vivo and following IVS. A harmonised IVS protocol was designed based on the best-performing protocol(s), which was then evaluated in a second phase on 2 donors by 6 centres. All centres were able to reliably detect antigen-specific T cells of high/intermediate frequency both ex vivo (Phase I) and post-IVS (Phase I and II). The highest frequencies of antigen-specific T cells ex vivo were mirrored in the frequencies following IVS and in the detection rates. However, antigen-specific T cells of a low/undetectable frequency ex vivo were not reproducibly detected post-IVS. Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %. We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R (2) = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation

Human bronchial fibroblasts express the 5-lipoxygenase pathway.

BACKGROUND: Fibroblasts are implicated in sub-epithelial fibrosis in remodeled asthmatic airways and contribute to airway inflammation by releasing cytokines and other mediators. Fibroblast activity is influenced by members of the leukotriene family of bronchoconstrictor and inflammatory mediators, but it is not known whether human bronchial fibroblasts can synthesize leukotrienes. METHODS: The expression of leukotriene biosynthetic enzymes and receptors was investigated in primary fibroblasts from the bronchi of normal and asthmatic adult subjects using RT-PCR, Western blotting, immunocytochemistry and flow cytometry. RESULTS: These techniques revealed that human bronchial fibroblasts from both subject groups constitutively express 5-lipoxygenase, its activating protein FLAP, the terminal enzymes leukotriene A4 hydrolase and leukotriene C4 synthase, and receptors for leukotriene B4 (BLT1) and cysteinyl-leukotrienes (CysLT1). Human bronchial fibroblasts generated immunoreactive leukotriene B4 and cysteinyl-leukotrienes spontaneously and in increased amounts after calcium-dependent activation. Flow cytometry showed that human bronchial fibroblasts transformed to a myofibroblast-like phenotype by culture with transforming growth factor-beta1 expressed 320-400% more immunofluorescence for leukotriene C4 synthase and CysLT1 receptors, with 60-80% reductions in leukotriene A4 hydrolase and BLT1 receptors. CONCLUSION: These results indicate that human bronchial fibroblasts may not only respond to exogenous leukotrienes but also generate leukotrienes implicated in narrowing, inflammation and remodeling of the asthmatic airway

Human bronchial epithelial cells express an active and inducible biosynthetic pathway for leukotrienes B4 and C4

BACKGROUND: Human bronchial epithelial cells synthesize cyclooxygenase and 15-lipoxygenase products, but the 5-lipoxygenase (5-LO) pathway that generates the leukotriene (LT) family of bronchoconstrictor and pro-inflammatory mediators is thought to be restricted to leucocytes. OBJECTIVE: We hypothesized that human bronchial epithelial cells (HBECs) express a complete and active 5-LO pathway for the synthesis of LTB4 and LTC4, either constitutively or after stimulation. METHODS: Flow cytometry, RT-PCR, Western blotting, enzyme immunoassays and reverse-phase high-performance liquid chromatography were used to investigate constitutive and stimulated expression of 5-LO pathway enzymes and the synthesis of LTs B4 and C4 in primary HBECs and in the 16-HBE 14o- cell line. RESULTS: Constitutive mRNA and protein expression for 5-LO, 5-LO-activating protein (FLAP), LTA4 hydrolase and LTC4 synthase were demonstrated in primary HBECs and in the 16-HBE 14o- cell line. In 16-HBE 14o- cells, treatment with calcium ionophore A23187, bradykinin or LPS up-regulated the expression of these enzymes. The up-regulation of 5-LO was blocked by the anti-inflammatory glucocorticoid dexamethasone. Human bronchial epithelial cells were shown to generate bioactive LTs, with primary HBECs generating 11-fold more LTC4 and five-fold more LTB4 than 16-HBE 14o- cells. LT production was enhanced by ionophore treatment and blocked by the FLAP inhibitor MK-886. CONCLUSIONS: Expression of an active and inducible 5-LO pathway in HBEC suggests that damaged or inflamed bronchial epithelium may synthesize LTs that contribute directly to bronchoconstriction and leucocytosis in airway inflammation