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 A(4 )hydrolase and leukotriene C(4 )synthase, and receptors for leukotriene B(4 )(BLT1) and cysteinyl-leukotrienes (CysLT(1)). Human bronchial fibroblasts generated immunoreactive leukotriene B(4 )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-β(1 )expressed 320–400% more immunofluorescence for leukotriene C(4 )synthase and CysLT(1 )receptors, with 60–80% reductions in leukotriene A(4 )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

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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00262-014-1593-0) contains supplementary material, which is available to authorized users

Colonic expression of leukotriene-pathway enzymes in inflammatory bowel diseases

BACKGROUND: Leukotrienes derived from the 5-lipoxygenase pathway are proinflammatory lipid mediators that possibly play a role in inflammatory bowel diseases. The expression of 5-lipoxygenase pathway proteins has not previously been examined in colonic mucosa in inflammatory bowel disease. RESULTS: Quantitative immunohistochemical analyses showed that, compared to those of the control subjects (n = 9), colonic biopsies from patients with active inflammatory bowel disease (n = 17) had 3- to 7-fold higher mean counts of cells expressing 5-lipoxygenase (P = 0.03), 5-lipoxygenase-activating protein (P = 0.005), and the leukotriene A(4) hydrolase (P = 0.004), which make up the biosynthetic pathway of the potent neutrophil chemotaxin leukotriene B(4). Immunoexpression of the leukotriene C(4) synthase was unaltered (P &gt; 0.2). The increased representation of leukotriene B(4)-pathway enzymes was associated with higher counts of neutrophils (P = 0.0001), macrophages (P = 0.03), eosinophils (P = 0.0004), CD8(+) T cells (P 0.9). These eicosanoid and cellular changes were most marked in the subgroup of patients with ulcerative colitis (n = 9), and were absent in patients with quiescent disease (n = 6). The anomalies in the 5-lipoxygenase pathway were accompanied as expected by more cells immunostaining for cytokine-inducible COX-2 (P = 0.004, n = 17), but this study also revealed a greater number of cells expressing COX-1 in the samples from the patients in the ulcerative colitis subgroup (P = 0.03, n = 9). CONCLUSIONS: The 5-lipoxygenase data provide a cellular basis for increased tissue synthesis of the leukotriene B(4), as reflected in the colonic mucosa and rectal dialysates of patients with active inflammatory bowel disease, which contributes to neutrophil influx and colonic injury. The COX-1/COX-2 data highlight the ambiguous functional role of prostanoid pathways in inflammatory bowel diseases

M1(hot)tumor-associated macrophages boost tissue-resident memory T cells infiltration and survival in human lung cancer

Background: the role of Tumour-Associated Macrophages (TAMs) in determining the outcome between the anti-tumour effects of the adaptive immune system and the tumour’s anti-immunity stratagems, is controversial. Macrophages modulate their activities and phenotypes by integration of signals in the tumour micro-environment. Depending on how macrophages are activated, they may adopt so-called M1-like, anti-tumour or M2-like, pro-tumour profiles. In many solid tumours, a dominance of M2-like macrophages is associated with poor outcomes but in some tumour types, strong M1-like profiles are linked to better outcomes. We aimed to investigate the inter-relationship of these TAM populations to establish how they modulate the efficacy of the adaptive immune system in early lung cancer.Methods: macrophages from matched lung (NTAMs) and tumour samples (TAMs) from resected lung cancers were assessed by bulk and single-cell transcriptomic analysis. Protein expression of genes characteristic of M1-like (CXCL9) or M2-like (MMP12) functions was confirmed by confocal microscopy. Immunohistochemistry related the distribution of TAM transcriptomic signatures to density of CD8+ tissue-resident memory T cells (TRM) in tumours and survival data from an independent cohort of 393 lung cancer patientsResults: TAMs have significantly different transcriptomic profiles from NTAMs with &gt;1000 differentially expressed genes. TAMs displayed a strong M2-like signature with no significant variation between patients. However, single-cell RNA-seq supported by immuno-stained cells revealed that additionally, in 25% of patients the M2-like TAMs also co-expressed a strong/hot M1-like signature (M1hot). Importantly, there was a strong association between the density of M1hot TAMs and TRM cells in tumours that was in turn linked to better survival. Our data suggests a mechanism by which M1hot TAMs may recruit TRM cells via CXCL9 expression and sustain them by making available more of the essential fatty acids on which TRM depend.Conclusions: we showed that in early lung cancer, expression of M1-like and M2-like gene signatures are not mutually exclusive since the same TAMs can simultaneously display both gene-expression profiles. The presence of M1hot TAMs was associated with a strong TRM tumour-infiltrate and better outcomes. Thus, therapeutic approaches to re-program TAMs to an M1hot phenotype are likely to augment the adaptive anti-tumour responses

Targeting carcinoembryonic antigen with DNA vaccination: on-target adverse events link with immunological and clinical outcomes

PURPOSE: We have clinically evaluated a DNA fusion vaccine to target the HLA-A*0201 binding peptide CAP-1 from carcinoembryonic antigen (CEA605-613) linked to an immunostimulatory domain (DOM) from fragment C of tetanus toxin Experimental Design: Twenty-seven patients with CEA-expressing carcinomas were recruited: 15 patients with measurable disease (Arm-I) and 12 patients without radiological evidence of disease (Arm-II). Six intramuscular vaccinations of naked DNA (1mg/dose) were administered up to week 12. Clinical and immunological follow-up was to week 64 or clinical/radiological disease.RESULTS: DOM-specific immune responses demonstrated successful vaccine delivery. All patients without measurable disease compared to 60% with advanced disease responded immunologically, while 58% and 20% expanded anti-CAP-1 CD8+ T-cells, respectively. CAP-1-specific T-cells were only detectable in the blood post-vaccination, but could also be identified in previously resected cancer tissue. The gastrointestinal adverse event diarrhea was reported by 48% of patients and linked to more frequent decreases in CEA (p&lt;0.001) and improved global immunological responses (anti-DOM responses of greater magnitude (p&lt;0.001), frequency (p=0.004) and duration) compared to patients without diarrhea. In advanced disease patients, decreases in CEA were associated with better overall survival (HR=0.14, p=0.017). CAP-1 peptide was detectable on MHC class I of normal bowel mucosa and primary colorectal cancer tissue by mass-spectrometry, offering a mechanistic explanation for diarrhea through CD8+ T-cell attack.CONCLUSIONS: Our data suggest that DNA vaccination is able to overcome peripheral tolerance in normal and tumor tissue and warrants testing in combination studies, for example, by vaccinating in parallel to treatment with an anti-PD1 antibody