Quantitative analysis of the CD4+ T cell response to therapeutic antibodies in healthy donors using a novel T cell:PBMC assay

Many biopharmaceuticals (BPs) are known to be immunogenic in the clinic, which can result in modified pharmacokinetics, reduced efficacy, allergic reactions and anaphylaxis. During recent years, several technologies to predict immunogenicity have been introduced, but the predictive value is still considered low. Thus, there is an unmet medical need for optimization of such technologies. The generation of T cell dependent high affinity anti-drug antibodies plays a key role in clinical immunogenicity. This study aimed at developing and evaluating a novel in vitro T cell:PBMC assay for prediction of the immunogenicity potential of BPs. To this end, we assessed the ability of infliximab (anti-TNF-α), rituximab (anti-CD20), adalimumab (anti-TNF-α) and natalizumab (anti-α4-integrin), all showing immunogenicity in the clinic, to induce a CD4+ T cells response. Keyhole limpet hemocyanin (KLH) and cytomegalovirus pp65 protein (CMV) were included as neo-antigen and recall antigen positive controls, respectively. By analyzing 26 healthy donors having HLA-DRB1 alleles matching the European population, we calculated the frequency of responding donors, the magnitude of the response, and the frequency of BP-specific T cells, as measured by 3[H]-thymidine incorporation and ELISpot IL-2 secretion. KLH and CMV demonstrated a strong T cell response in all the donors analyzed. The frequency of responding donors to the BPs was 4% for infliximab, 8% for adalimumab, 19% for rituximab and 27% for natalizumab, which is compared to and discussed with their respective observed clinical immunogenicity. This study further complements predictive immunogenicity testing by quantifying the in vitro CD4+ T cell responses to different BPs. Even though the data generated using this modified method does not directly translate to the clinical situation, a high sensitivity and immunogenic potential of most BPs is demonstrated

Diminished levels of nasal S100A7 (psoriasin) in seasonal allergic rhinitis: an effect mediated by Th2 cytokines

<p>Abstract</p> <p>Background</p> <p>S100A7 is an antimicrobial peptide involved in several inflammatory diseases. The aim of the present study was to explore the expression and regulation of S100A7 in seasonal allergic rhinitis (SAR).</p> <p>Methods</p> <p>Nasal lavage (NAL) fluid was obtained from healthy controls before and after lipopolysaccharide (LPS) provocation, from SAR patients before and after allergen challenge, and from SAR patients having completed allergen-specific immunotherapy (ASIT). Nasal biopsies, nasal epithelial cells and blood were acquired from healthy donors. The airway epithelial cell line FaDu was used for <it>in vitro </it>experiments. Real-time RT-PCR and immunohistochemistry were used to determine S100A7 expression in nasal tissue and cells. Release of S100A7 in NAL and culture supernatants was measured by ELISA. The function of recombinant S100A7 was explored in epithelial cells, neutrophils and peripheral blood mononuclear cells (PBMC).</p> <p>Results</p> <p>Nasal administration of LPS induced S100A7 release in healthy non-allergic subjects. The level of S100A7 was lower in NAL from SAR patients than from healthy controls, and it was further reduced in the SAR group 6 h post allergen provocation. In contrast, ASIT patients displayed higher levels after completed treatment. S100A7 was expressed in the nasal epithelium and in glands, and it was secreted by cultured epithelial cells. Stimulation with IL-4 and histamine repressed the epithelial S100A7 release. Further, recombinant S100A7 induced activation of neutrophils and PBMC.</p> <p>Conclusions</p> <p>The present study shows an epithelial expression and excretion of S100A7 in the nose after microbial stimulation. The levels are diminished in rhinitis patients and in the presence of an allergic cytokine milieu, suggesting that the antimicrobial defense is compromised in patients with SAR.</p

The Innate Immune Cross Talk between NK Cells and Eosinophils Is Regulated by the Interaction of Natural Cytotoxicity Receptors with Eosinophil Surface Ligands

Previous studies suggested that the cross talk between NK cells and other cell types is crucial for the regulation of both innate and adaptive immune responses. In the present study, we analyzed the phenotypic and functional outcome of the interaction between resting or cytokine-activated NK cells and eosinophils derived from non-atopic donors. Our results provide the first evidence that a natural cytotoxicity receptor (NCR)/NCR ligand-dependent cross talk between NK cells and eosinophils may be important to upregulate the activation state and the effector function of cytokine-primed NK cells. This interaction also promotes the NK-mediated editing process of dendritic cells that influence the process of Th1 polarization. In turn, this cross talk also resulted in eosinophil activation and acquisition of the characteristic features of antigen-presenting cells. At higher NK/eosinophil ratios, cytokine-primed NK cells were found to kill eosinophils via NKp46 and NKp30, thus suggesting a potential immunoregulatory role for NK cells in dampening inflammatory responses involving eosinophils

Upregulated levels of human β-defensins in patients with seasonal allergic rhinitis after allergen-specific immunotherapy treatment.

BACKGROUND: Antimicrobial peptides (AMPs) are important actors in the innate immune system. One class of AMPs is the human β-defensins (HBDs), a group of small peptides with a broad spectrum of antimicrobial activities. Expression of HBDs is downregulated in different manifestations of allergic disease. In this study, we examine whether allergen-specific immunotherapy (ASIT) affects the nasal levels of HBDs in patients with seasonal allergic rhinitis (SAR). METHODS: Nasal biopsies were examined for the occurrence of HBD1-3 with real-time reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. Nasal lavage (NAL) fluids from healthy individuals, untreated SAR patients and SAR patients before and after ASIT were analyzed for levels of HBD1-3 using enzyme-linked immunosorbent assay (ELISA). RESULTS: Examination of nasal biopsies revealed HBD1-3 expression at gene level as well as at protein level in all samples tested. HBD1 and HBD3 messenger RNA (mRNA) levels were downregulated in SAR patients compared to healthy individuals. All HBDs were found in NAL fluids. SAR patients having completed 3 years of ASIT displayed higher levels of HBD1 and HBD2 than before treatment, whereas levels of HBD3 were unaffected. CONCLUSION: The present study demonstrates an upregulation of HBD1 and HBD2 in SAR patients after completion of ASIT. This may reflect the importance of an intact innate immune response as part of our defense against infections among allergic individuals

Innate immune receptors in human airway smooth muscle cells: activation by TLR1/2, TLR3, TLR4, TLR7 and NOD1 agonists.

BACKGROUND: Pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs), recognize microbial components and trigger a host defense response. Respiratory tract infections are common causes of asthma exacerbations, suggesting a role for PRRs in this process. The present study aimed to examine the expression and function of PRRs on human airway smooth muscle cells (HASMCs). METHODS: Expression of TLR, NLR and RLR mRNA and proteins was determined using real-time RT-PCR, flow cytometry and immunocytochemistry. The functional responses to ligand stimulation were investigated in terms of cytokine and chemokine release, cell surface marker expression, proliferation and proteins regulating the contractile state. RESULTS: HASMCs expressed functional TLR2, TLR3, TLR4, TLR7 and NOD1. Stimulation with the corresponding agonists Pam3CSK4, poly(I:C), LPS, R-837 and iE-DAP, respectively, induced IL-6, IL-8 and GM-CSF release and up-regulation of ICAM-1 and HLA-DR, while poly(I:C) also affected the release of eotaxin and RANTES. The proliferative response was slightly increased by LPS. Stimulation, most prominently with poly(I:C), down-regulated myosin light chain kinase and cysteinyl leukotriene 1 receptor expression and up-regulated β2-adrenoceptor expression. No effects were seen for agonist to TLR2/6, TLR5, TLR8, TLR9, NOD2 or RIG-I/MDA-5. CONCLUSION: Activation of TLR2, TLR3, TLR4, TLR7 and NOD1 favors a synthetic phenotype, characterized by an increased ability to release inflammatory mediators, acquire immunomodulatory properties by recruiting and interacting with other cells, and reduce the contractile state. The PRRs might therefore be of therapeutic use in the management of asthma and infection-induced disease exacerbations

Functional Effects of Toll-Like Receptor (TLR)3, 7, 9, RIG-I and MDA-5 Stimulation in Nasal Epithelial Cells.

The human nasal epithelium is an important physical barrier, and a part of the innate immune defense that protect against pathogens. The epithelial cells recognize microbial components by pattern-recognition receptors (PRRs), and thereby trigger an immune response. Even though TLR3, TLR7, TLR9, RIG-I and MDA-5 are all known to respond to viral stimulation, their potential role in chronic airway inflammation triggered by local cytokine release remains to be established

Expression of TLR2, TLR3, TLR4, TLR7 and NOD1 proteins.

<p>(<b>A–E</b>) HASMCs were stained intracellularly with Abs against TLR2, TLR3, TLR4, TLR7 and NOD1 (open histograms) or appropriate isotype control (shaded histograms) and analyzed by flow cytometry. Data show one out of three independent experiments. (<b>F</b>) Data are presented as relative mean fluorescence intensity (rMFI = MFI_Ab/MFI_{isotype control}) and depicted as mean ± SEM (n = 3). (<b>G</b>) Immunocytochemistry of HASMCs stained with Abs against TLR2, (<b>H</b>) TLR3 (diluted 1∶10), (<b>I</b>) TLR4, (<b>J</b>) TLR7, (<b>K</b>) NOD1 (diluted 1∶50), and (<b>L</b>) N-series negative control reagent (diluted 1∶10). Slides were visualized using 3,3′-diaminobenzidine (brown). All slides were counterstained with hematoxylin (blue) and analyzed by microscopy; magnification 200×.</p

Nod-like receptors in head and neck squamous cell carcinoma

Conclusion: The capability of Nod1 to recognize bacteria along with its altered expression and ability to cause an immunological response in head and neck cancer suggest a novel pathway for bacteria to interfere with ongoing cancer inflammation. Objective: Nucleotide oligomerization domain (Nod)-like receptors (NLRs) comprise a recently discovered family of pattern-recognition receptors. In addition to their protective function against infections, accumulating evidence suggests a role for these receptors in various diseases, including cancer. The present study was designed to explore the presence of NLRs in head and neck squamous cell carcinoma, and to determine if these cells have the ability to respond immunologically to ligand stimulation. Methods: The pharyngeal squamous cell carcinoma cell lines Detroit-562 and FaDu were used as a model for head and neck cancer, and compared to healthy primary human nasal epithelial cells. Analyses were performed using immuno-histochemistry, real-time RT-PCR, Luminex Multiplex Immunoassay, ELISA, and flow cytometry. Results: The expression profile of NLRs in head and neck cancer cells differed from that seen in healthy epithelial cells. Further, Nod1 stimulation induced an immunological response in tumor cells that differed from the response in normal epithelial cells, especially regarding the expression of beta-defensin 2, granulocyte monocyte colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), intercellular adhesion molecule-1 (ICAM-1), and cell survival

The activation pattern of blood leukocytes in head and neck squamous cell carcinoma is correlated to survival.

Head and neck squamous cell carcinoma (HNSCC) is known to cause substantial immunosuppression. The present study was designed to characterize blood leukocyte activation in HNSCC and to investigate if the individual activation pattern could be related to tumor progress and survival. The leukocyte activation profile of HNSCC patients and healthy controls was assessed with flow cytometry. HNSCC patients displayed increased numbers of monocytes, neutrophils and total leukocytes as well as an enhanced neutrophil/lymphocyte ratio. In addition, patients had a higher percentage of CD69(+), CD71(+) and CD98(+) T cell subsets and NK cells, and a reduced expression of L-selectin in CD14(high)CD16(+) monocytes and neutrophils, when compared to controls. These changes could be correlated to both tumor burden and spread to lymph nodes. Among the cancer patients an increased neutrophil/lymphocyte ratio, a low neutrophil and CD14(high) CD16(+) monocyte activation state and an elevated CD4/CD8 ratio were related to poor survival. In contrast, a high percentage of CD98(+) Th cells appeared to be associated with a better outcome. Taken together, the present data indicate that HNSCC causes activation of blood leukocytes and that the individual activation pattern can be linked to prognosis

The TLR7 antagonist IRS661 completely abolishes the effect induced by R-837.

<p>HASMCs were pretreated for 1 h with IRS661 (0.175 µM) prior to the addition of R-837 (10 µM). After 24 h, the cell-free culture supernatants were recovered and analyzed for levels of IL-6 by ELISA. Data are presented as mean ± SEM (n = 6). *, p<0.05.</p