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

Wilms’ tumour antigen 1 Immunity via DNA fusion gene vaccination in haematological malignancies by intramuscular injection followed by intramuscular electroporation: a Phase II non-randomised clinical trial (WIN)

Background: In the UK almost 7000 people are diagnosed with leukaemia each year, but despite continuing advances in diagnosis and treatment with new drugs, such as the tyrosine kinase inhibitors, the majority of these patients will eventually die from their disease. Until quite recently, the only treatment to offer the possibility of long-term disease-free survival was allogeneic stem cell transplantation. However, this carries a substantial risk of mortality and is available to only a minority of patients.Objectives: The aim of the study was to test the hypothesis that molecular and clinical responses, induced by T lymphocytes (T cells), can be predicted by increases in the number of CD8+ (cluster of differentiation 8-positive) T cells specific for the vaccine-encoded T-cell epitopes. This project also aimed to build on the established programme of deoxyribonucleic acid (DNA) fusion-gene vaccination delivered by intramuscular injection, exploiting a unique experience with electroporation, to induce durable immune responses with the aim of controlling disease by precision attack of the tumour by CD8+ T cells.Method: A non-randomised, open-label, single-dose-level Phase II clinical trial in two patient groups [chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML)] on stable doses of imatinib. Human leucocyte antigen A2-positive (HLA A2+) patients were vaccinated with two DNA vaccines: (1) p.DOM–WT1-37 (epitope sequence: VLDFAPPGA); and (2) p.DOM–WT1-126 (epitope sequence: RMFPNAPYL). The HLA A2-negative patients formed an unvaccinated control group. The sample size for the HLA A2+ group was originally determined following Simon’s optimal Phase II trial design (Simon R. Optimal two-stage designs for phase II clinical trials. Control Clin Trials 1989;10:1–10). This was changed to A’Hern’s single-stage design during the course of the trial (A’Hern RP. Sample size tables for single-stage phase II designs. Stat Med 2001;20:859–66), which was endorsed by the trial’s independent oversight committees.Results: The study included 12 patients with CML who were vaccinated and nine patients with CML who were unvaccinated as the control group. Both the vaccines and the electroporation were safe, with no new or unexpected toxicities. The evaluation adverse events of special interest (heart, bone marrow, renal) did not reveal safety concerns. Two BCR–ABL (breakpoint cluster region–Abelson murine leukaemia viral oncogene homolog 1) responses were observed, both of which were defined as a major response, with one in each group. Two Wilms’ tumour antigen 1 (WT1) molecular responses were observed in the vaccinated group and one was observed in the control group. At an immunological level, the vaccine performed as expected.Conclusions: The study met its primary decision-making target with one major molecular response in BCR–ABL transcript levels. Overall, the data showed, in this clinical setting, the immunogenicity and safety of the vaccine.Limitations: The study did not complete recruitment and there were multiple hurdles that contributed to this failure. This is disappointing given the robust induction immune responses against WT1 T-cell responses in 7 out of 10 evaluable patients.Future work: Evaluation of the p.DOM–WT1 vaccines in AML remains attractive clinically, but it is unlikely to be feasible at this time. Combination of the DNA vaccine approach with strategies to expand T-cell responses with immunomodulatory antibodies is in development.Funding details: This project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a Medical Research Council (MRC) and National Institute for Health Research (NIHR) partnership, and Bloodwise

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

Modification of dendritic cell phenotype and function : consequences for allergic asthma

The initial work aimed to characterise monocyte-derived dendritic cells (Mo-DCs) from normal and asthmatic subjects to identify inherited differences with possible relevance for allergic asthma. It was found that Mo-DCs from asthmatic subjects expressed significantly lower level of CD23 at their surface and produced more IL-6 than Mo-DCs from normal subjects.A primary screen was then developed to address the hypothesis. The primary screen aimed to identify individual agents found at elevated levels in the asthmatic lung, referred to as allergic mediators, that significantly change the phenotype or function of Mo-DCs. Further in-depth investigation of their effects on Mo-DCs and possible relevance in allergic asthma would then be conducted. The effects of allergic mediators on Mo-DCs from both normal and asthmatic subjects were studied. The experimental approach taken showed that the Mo-DC phenotype and function was significantly changed in response to TNF-α (the positive control), IFN-γ, IL-3, IL-5 and IgE, but not in response to MMIP-1α, histamine, PGD2 and IL-13. Four areas of particular interest were identified. 1) Phenotypically mature IFN-γ treated Mo-DCs failed to significantly enhance T cell proliferation. 2) CD23 disappeared from the surface of Mo-DCs in response to TNF-α, IFN-γ and IgE. 3) IL-3 and IL-5 frequently induced a mature Mo-DC phenotype. 4) Mo-DCs from normal and asthmatic subjects responded differently to allergic mediators.The work presented in this thesis supports a role for DCs in allergic asthma. Evidence also suggested that DCs from asthmatic subjects are more prone to initiate or perpetuate the chronic allergic inflammation seen in asthmatic airways.</p

Wilms’ tumour antigen 1 Immunity via DNA fusion gene vaccination in haematological malignancies by intramuscular injection followed by intramuscular electroporation: a Phase II non-randomised clinical trial (WIN)

Background: In the UK almost 7000 people are diagnosed with leukaemia each year, but despite continuing advances in diagnosis and treatment with new drugs, such as the tyrosine kinase inhibitors, the majority of these patients will eventually die from their disease. Until quite recently, the only treatment to offer the possibility of long-term disease-free survival was allogeneic stem cell transplantation. However, this carries a substantial risk of mortality and is available to only a minority of patients. Objectives: The aim of the study was to test the hypothesis that molecular and clinical responses, induced by T lymphocytes (T cells), can be predicted by increases in the number of CD8+ (cluster of differentiation 8-positive) T cells specific for the vaccine-encoded T-cell epitopes. This project also aimed to build on the established programme of deoxyribonucleic acid (DNA) fusion-gene vaccination delivered by intramuscular injection, exploiting a unique experience with electroporation, to induce durable immune responses with the aim of controlling disease by precision attack of the tumour by CD8+ T cells. Method: A non-randomised, open-label, single-dose-level Phase II clinical trial in two patient groups [chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML)] on stable doses of imatinib. Human leucocyte antigen A2-positive (HLA A2+) patients were vaccinated with two DNA vaccines: (1) p.DOM–WT1-37 (epitope sequence: VLDFAPPGA); and (2) p.DOM–WT1-126 (epitope sequence: RMFPNAPYL). The HLA A2-negative patients formed an unvaccinated control group. The sample size for the HLA A2+ group was originally determined following Simon’s optimal Phase II trial design (Simon R. Optimal two-stage designs for phase II clinical trials. Control Clin Trials 1989;10:1–10). This was changed to A’Hern’s single-stage design during the course of the trial (A’Hern RP. Sample size tables for single-stage phase II designs. Stat Med 2001;20:859–66), which was endorsed by the trial’s independent oversight committees. Results: The study included 12 patients with CML who were vaccinated and nine patients with CML who were unvaccinated as the control group. Both the vaccines and the electroporation were safe, with no new or unexpected toxicities. The evaluation adverse events of special interest (heart, bone marrow, renal) did not reveal safety concerns. Two BCR–ABL (breakpoint cluster region–Abelson murine leukaemia viral oncogene homolog 1) responses were observed, both of which were defined as a major response, with one in each group. Two Wilms’ tumour antigen 1 (WT1) molecular responses were observed in the vaccinated group and one was observed in the control group. At an immunological level, the vaccine performed as expected. Conclusions: The study met its primary decision-making target with one major molecular response in BCR–ABL transcript levels. Overall, the data showed, in this clinical setting, the immunogenicity and safety of the vaccine. Limitations: The study did not complete recruitment and there were multiple hurdles that contributed to this failure. This is disappointing given the robust induction immune responses against WT1 T-cell responses in 7 out of 10 evaluable patients. Future work: Evaluation of the p.DOM–WT1 vaccines in AML remains attractive clinically, but it is unlikely to be feasible at this time. Combination of the DNA vaccine approach with strategies to expand T-cell responses with immunomodulatory antibodies is in development. Funding details: This project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a Medical Research Council (MRC) and National Institute for Health Research (NIHR) partnership, and Bloodwise

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