Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4⁺ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4⁺ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17^- and CCL22-induced movement of CCR4⁺ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 µm pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

The airway inflammatory response is closely associated with asthma. The purpose of this article was to study the roles of innate lymphoid cells (ILCs) in the process of airway inflammatory response in asthma. We established the asthmatic mice model with intraperitoneal injected ovalbumin medium, then with the flow cytometry analysis, we detected the ILCs and their surface proteins in the mice blood samples, besides, we analyzed the amounts of inflammatory cytokines and secreted proteins in the mice bronchoalveolar lavage fluid and blood serum. Moreover, Western blot analyzed the proteins in the mice bronchial epithelial tissues. The ILC2 amounts were obviously increased in young asthmatic mice model. And, the proteins CD25 and CCR10 were highly expressed in the sorted ILC2s. Besides, the cytokines interleukin (IL)-5, IL-13, IL-33, CCL22, and CCL27 were abundant in the bronchoalveolar lavage fluid of asthmatic mice model. And, the secretion of IL-5, IL-13, IL-33, TSLP, and CCL22 in blood serum was much more in asthmatic mice model than in the normal control mice, whereas the secretion of PGD2 was suppressed in asthmatic mice bronchoalveolar lavage fluid and blood serum. Additionally, the guanine nucleotide-binding proteins Gα12 and Gα13 were upregulated in asthmatic mice bronchial tissues, and the protein SERCA2 was downregulated; moreover, the proteins NFAT, IRF4, and its downstream signal STAT6 were all upregulated in the asthmatic mice bronchial tissues. ILC2s were involved in the response of airway inflammation through secretion of proinflammatory cytokines and chemokines to dysregulate the Ca²⁺ homeostasis in airway in the process of asthma.

Background and purpose: Epidemiological and experimental studies suggest that microbial exposure in early childhood is linked with reduced risk to suffer asthma and thus microbial components with immunoregulatory capabilities might serve as a novel preventive strategy for allergic asthma. Recently, it was identified that Streptococcus pneumoniae aminopeptidase N (PepN) could suppress T cell effector function. We sought to investigate the effect of PepN on murine allergic asthma and elucidate the underlying mechanism.

Experimental approach: The effects of intranasal administration of PepN during or before sensitization were examined in ovalbumin (OVA)-induced murine allergic asthma. The roles of CD11b⁺ dendritic cells in PepN treated OVA-induced allergic asthma were evaluated by flow cytometry, cytokines detection and adoptive transfer. Moreover, the numbers of lung type 2 innate lymphoid cells (ILC2s) were also detected.

Key results: Administration of PepN during or before sensitization attenuated type-2 airway inflammation (eosinophilia, mucus hypersecretion, Th2 cytokines production, IgE production) in allergic asthma mice. PepN reduced lung accumulation of CD11b⁺ DCs, which was accompanied by diminished DC-attracting chemokine CCL20 pruduction, as well as CCL17 and CCL22 which are Th2-cell chemokines predominantly produced by CD11b⁺ DCs. Adoptive transfer of BM-derived CD11b⁺ DCs abolished the inhibitory effect of PepN on OVA-induced type-2 airway inflammation. The numbers of lung ILC2s were decreased in asthmatic mice receving PepN.

Conclusions and implications: These results demonstrated that PepN alleviated type-2 inflammation in OVA-induced allergic asthma mice, which was mediated by regulation of lung CD11b⁺ DCs. Our study provides a novel strategy for the prevention of allergic asthma.

Keywords: Streptococcus pneumoniae aminopeptidase N; allergic asthma; dendritic cells; type 2 innate lymphoid cells.

Lignosus rhinocerotis Cooke. (L. rhinocerotis) is a medicinal mushroom traditionally used in the treatment of asthma and several other diseases by the indigenous communities in Malaysia. In this study, the effects of L. rhinocerotis on allergic airway inflammation and hyperresponsiveness were investigated. L. rhinocerotis extract (LRE) was prepared by hot water extraction using soxhlet. Airway hyperresponsiveness (AHR) study was performed in house dust mite (HDM)-induced asthma in Balb/c mice while airway inflammation study was performed in ovalbumin (OVA)-induced asthma in Sprague-Dawley rats. Treatment with different doses of LRE (125, 250 and 500 mg/kg) significantly inhibited AHR in HDM-induced mice. Treatment with LRE also significantly decreased the elevated IgE in serum, Th2 cytokines in bronchoalveolar lavage fluid and ameliorated OVA-induced histological changes in rats by attenuating leukocyte infiltration, mucus hypersecretion and goblet cell hyperplasia in the lungs. LRE also significantly reduced the number of eosinophils and neutrophils in BALF. Interestingly, a significant reduction of the FOXP3+ regulatory T lymphocytes was observed following OVA induction, but the cells were significantly elevated with LRE treatment. Subsequent analyses on gene expression revealed regulation of several important genes i.e. IL17A, ADAM33, CCL5, IL4, CCR3, CCR8, PMCH, CCL22, IFNG, CCL17, CCR4, PRG2, FCER1A, CLCA1, CHIA and Cma1 which were up-regulated following OVA induction but down-regulated following treatment with LRE. In conclusion, LRE alleviates allergy airway inflammation and hyperresponsiveness, thus suggesting its therapeutic potential as a new armamentarium against allergic asthma.

Background: Chronic obstructive pulmonary disease (COPD) is commonly associated with both a pro-inflammatory and a T-helper 1 (Th1) immune response. It was hypothesized that cannabis oil extract can alleviate COPD symptoms by eliciting an anti-inflammatory Th2 immune response. Accordingly, the effects of cannabis oil extract on the expression of 84 Th2 and related immune response genes in human small airways epithelial cells (HSAEpC) were investigated.

Methods: HSAEpC from a single donor were treated with three dilutions of a standardized cannabis oil extract (1:400, 1:800 and 1:1600) along with a solvent control (0.25% [2.5 ul/ml] ethanol) for 24 h. There were four replicates per treatment dilution, and six for the control. RNA isolated from cells were employed in pathway-focused quantitative polymerase chain reaction (qPCR) microarray assays.

Results: The extract induced significant (P < 0.05) changes in expression of 37 tested genes. Six genes (CSF2, IL1RL1, IL4, IL13RA2, IL17A and PPARG) were up-regulated at all three dilutions. Another two (CCL22 and TSLP) were up-regulated while six (CLCA1, CMA1, EPX, LTB4R, MAF and PMCH) were down-regulated at the 1:400 and 1:800 dilutions. The relationship of differentially-expressed genes of interest to biologic pathways was explored using the Database for Annotation, Visualization and Integrated Discovery (DAVID).

Conclusions: This exploratory investigation indicates that cannabis oil extract may affect expression of specific airway epithelial cell genes that could modulate pro-inflammatory or Th1 processes in COPD. These results provide a basis for further investigations and have prompted in vivo studies of the effects of cannabis oil extract on pulmonary function.

Trial registration: NONE (all in vitro experiments).

Keywords: Anti-inflammatory; Cannabis; Chronic obstructive pulmonary disease (COPD); Gene expression profiling; HSAEpC (human small airways epithelial cells); KEGG pathway analysis; Th1 and Th2 immune response.

BACKGROUND

Interleukin (IL) 16 and thymus and activation-regulated cytokine (TARC) are chemoattractant cytokines for eosinophils and TH2 cells. Differential levels of these components in aspirin-exacerbated respiratory disease (AERD) and allergic rhinitis with asthma (ARwA) may be related to a different inflammatory response in both asthma phenotypes.

OBJECTIVE

To assess the nasal lavage immunoreactivity of IL-16 and TARC cytokines.

METHODS

We used multienzyme-linked immunosorbent assays to detect IL-5, IL-13, IL-16, IL-33, I-309/CCL1, TARC/CCL17, monocyte-derived chemokine/CCL22, periostin, and eosinophil cationic protein levels in nasal lavages from patients with AERD and patients with ARwA.

RESULTS

The IL-13, IL-16, TARC, and periostin levels were significantly higher in patients with AERD compared with those of patients with ARwA. Correlation analysis of mediator levels in AERD revealed a possible role of IL-16 and TARC in eosinophil recruitment and activation.

CONCLUSIONS

IL-16, TARC, and periostin distinguish between patients with AERD and those with ARwA. These mediators, taken together rather than individually, may comprise good specific nasal markers in patients with AERD. The effects of IL-16 and TARC on TH1, TH2, and T-regulatory cell functions in AERD cannot be disregarded.

RNA-protein interactions with physiological outcomes usually rely on conserved sequences within the RNA element. By contrast, activity of the diverse gamma-interferon-activated inhibitor of translation (GAIT)-elements relies on the conserved RNA folding motifs rather than the conserved sequence motifs. These elements drive the translational silencing of a group of chemokine (CC/CXC) and chemokine receptor (CCR) mRNAs, thereby helping to resolve physiological inflammation. Despite sequence dissimilarity, these RNA elements adopt common secondary structures (as revealed by 2D-1H NMR spectroscopy), providing a basis for their interaction with the RNA-binding GAIT complex. However, many of these elements (e.g. those derived from CCL22, CXCL13, CCR4 and ceruloplasmin (Cp) mRNAs) have substantially different affinities for GAIT complex binding. Toeprinting analysis shows that different positions within the overall conserved GAIT element structure contribute to differential affinities of the GAIT protein complex towards the elements. Thus, heterogeneity of GAIT elements may provide hierarchical fine-tuning of the resolution of inflammation.

A key issue in the field of tissue engineering and stem cell therapy is immunological rejection after the implantation of allogeneic bone marrow-derived mesenchymal stem cells (BMSCs). In addition, maintaining the immunoregulatory function of BMSCs is critical to achieving tissue repair. In recent years, scientists have become interested in fish collagen because of its unique osteoinductive activity. However, it is still unclear whether osteogenically differentiated BMSCs induced by fish collagen maintain their immunoregulatory functions. To address this question, BMSCs were isolated from 8-week-old male BALB/c mice, and a noncontact coculture model was established consisting of macrophages and BMSCs treated with hydrolyzed fish collagen (HFC). Cell proliferation of the macrophages was determined by MTT. The gene and protein expression levels of the M1 and M2 macrophage markers were measured by real-time PCR and enzyme-linked immunosorbent assay (ELISA). To study the role of TNF-α-induced gene/protein 6 (TSG-6), TSG-6 was targeted by short interfering RNA (siRNA) in BMSCs, then the osteogenic differentiation ability of the BMSCs was examined by western blotting. The mRNA expression levels of interleukin-10 (IL-10), CCL22 (a macrophage-derived chemokine), tumor necrosis factor α (TNF-α), and interleukin-12 (IL-12), and the protein expression levels of arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS) of macrophages cocultured with TSG-6-siRNA-BMSCs+HFC were detected by real-time PCR and western blotting, respectively. The results showed that the osteogenically differentiated BMSCs induced by HFC did not affect the proliferation of macrophages. Osteogenically differentiated BMSCs induced by HFC promoted the expression of M2 macrophage markers IL-10 and CCL22, while HFC inhibited the expression of M1 macrophage markers, including TNF-α and IL-12. The TSG-6 knockdown led to a decrease in the production of TSG-6 without impairing the expression of bone sialoprotein (BSP), osteocalcin (OCN), and Runt-related transcription factor 2 (RUNX2) by BMSCs. TSG-6 silencing significantly counteracted the effect of HFC, and the expression of IL-10, CCL22, and Arg-1 were all decreased in the macrophages cocultured with TSG-6-siRNA-BMSCs+HFC, while that of TNF-α, IL-12, and iNOS were increased relative to the BMSCs+HFC group. The data demonstrated that osteogenically differentiated BMSCs induced by fish collagen retained their immunomodulatory functions. This study provides an additional scientific basis for future applications of fish collagen as an osteogenic component in the fields of tissue engineering and stem cell therapy.

In smoking-induced chronic obstructive pulmonary disease (COPD), various comorbidities are linked to systemic inflammation and infection-induced exacerbations. The underlying mechanisms are unclear but might provide therapeutic targets. T-cell activity is central in systemic inflammation and for infection-defense mechanisms and might be influenced by comorbidities. Hypothesis: Circulating biomarkers of comorbidities modulate the activity of T-cells of the T-helper type 1 (Th1) and/or T-cytotoxic type 1 (Tc1). T-cells in peripheral blood mononuclear cells (PBMCs) from non-smokers (NS), current smokers without COPD (S), and COPD subjects (total n = 34) were ex vivo activated towards Th1/Tc1 and were then stimulated with biomarkers for metabolic and/or cardiovascular comorbidities (Brain Natriuretic Peptide, BNP; chemokine (C-C motif) ligand 18, CCL18; C-X3-C motif chemokine ligand 1, CX3CL1; interleukin-18, IL-18) or for asthma- and/or cancer-related comorbidities (CCL22; epidermal growth factor, EGF; IL-17; periostin) each at 10 or 50 ng/mL. The Th1/Tc1 activation markers interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were analyzed in culture supernatants by Enzyme-Linked Immunosorbent Assay (ELISA). Ex-vivo activation induced IFNγ and TNFα without differences between the groups but GM-CSF more in S vs. NS. At 10 ng/mL, the different biomarkers increased or reduced the T-cell activation markers without a clear trend for one direction in the different categories of comorbidities or for the different T-cell activation markers. At 50 ng/mL, there was a clear shift towards suppressive effects, particularly for the asthma- and cancer-related biomarkers and in cells of S and COPD. Comorbidities might suppress T-cell immunity in COPD. This could explain the association of comorbidities with frequent exacerbations.

Keywords: COPD; T-cells; comorbidities.

BCC is an immunogenic tumor highlighted by the increased risk in immunosuppressed individuals and the frequent occurrence of tumor infiltrating lymphocytes (TILs) in the tumor surroundings. Immunotherapy is evolving as a promising treatment strategy for several cancer types where topical immunostimulators are among the possibilities for superficial BCC. The overall aim of this thesis is to characterize the immunologic response upon BCC as well as characterizing the surrounding tumor stroma. The aim was achieved by the use of a variety of laboratory techniques; immunohistochemistry, immunoflourescence, qRT-PCR and NGS. Tumor microenvironment: T-regs are a subpopulation of the CD4 positive T-cells normally comprising around 5-10% of the peripheral T-cells and up to 20% of the skin resident T-cells. In the healthy individual they are crucial in hindering autoimmune diseases whereas the role in cancer is less advantageous with association to tumor progression for a variety of cancer types. By investigating the presence of T-regs in BCC by immunohistochemistry in study I, it was found that T-regs comprised 45% in mean of the total CD4 positive cells in BCC. The increased T-reg concentration was confirmed with qRT-PCR showing increased Foxp3 expression levels in BCC as well as in the peritumoral skin. In the normal non-UV exposed buttock skin, no Foxp3 expression was found. Hence, T-regs seem to play a role both in BCC but also in the tumor surroundings. Tumor surroundings are essential in terms of the ability for a tumor to grow. Apart from interaction between immune and cancer cells, also crosstalk with cells of the connective tissue such as CAFs is essential. In study II, NGS revealed increased expression of the CAF-markers P4H and PDGFR-β in BCC. Subsequent qRT-PCR confirmed this and also showed increased expression in the peritumoral skin whereas no expression was found in the normal buttock skin. FAP-α expression was seen only within BCC. CAFs are thus highly present within BCC and we further hypothesize that fibroblasts in the peritumoral skin acquire a phenotype intermediate between normal fibroblasts and CAFs in BCC. This intermediate phenotype might be induced by chronic UV-exposure mediated by increased IL6 expression. This corresponds to our findings of highly increased IL6 expression primarily in the peritumoral skin and to previous literature describing CAF-induced tumor-promoting IL6 expression upon UV-exposure in cutaneous SCC. Recruitment of TILs to BCC: mRNA expression levels of the chemokines CCL17, CCL18, CCL22 and CXCL12, involved in T-reg attraction to tumor sites were increased both in tumor and peritumoral skin with lack of expression in the normal skin. Correlation between the chemokines CCL17 and CXCL12 and CAF markers was found by IF establishing a role for CAFs in attracting T-regs to tumor sites. Efficient immunologic anti-tumor response could be provided by clonal expansion of T-cells directed against tumor-antigens. If this was the case, then restricted TCR-repertoire in BCC compared with surrounding skin would be seen. Analysis of the α and β- chain of the TCR was performed showing a high diversity of TCR repertoire in BCC and lack of predominant V(D)J-gene usage, no preferential VJ pairing or specific CDR3 length distribution. Therefore, no support of antigen-driven clone selection was found. This corresponds with lack of obvious anti-tumor skewing towards a Th1, Th2 or Th17 polarization.

CONCLUSIONS

To summarize it has been shown, with these studies on the local immune response upon BCC development, that an immunologic response is generated in line with BCC being an immunogenic tumor. This response is not specific, though. Additionally, BCC is capable of generating a protective niche in the microenvironment composed of both T-regs and CAFs breeding local immunosuppression and hindering of adequate anti-tumor response. In a clinical perspective, further research in improving immunotherapy for BCC is promising since an immunological response is present but needs to be reactivated.

Recruitment of suppressive CD4⁺ FOXP3⁺ regulatory T cells (T_reg) to the tumor microenvironment (TME) has the potential to weaken the antitumor response in patients receiving treatment with immuno-oncology (IO) agents. Human T_reg express CCR4 and can be recruited to the TME through the CC chemokine ligands CCL17 and CCL22. In some cancers, T_reg accumulation correlates with poor patient prognosis. Preclinical data suggests that preventing the recruitment of T_reg and increasing the population of activated effector T cells (T_eff) in the TME can potentiate antitumor immune responses. We developed a novel series of potent, orally bioavailable small molecule antagonists of CCR4. From this series, several compounds exhibited high potency in distinct functional assays in addition to good in vitro and in vivo ADME properties. The design, synthesis, and SAR of this series and confirmation of its in vivo activity are reported.

Locally advanced rectal cancer is treated with neoadjuvant-chemoradiotherapy; however, only ~22% of patients achieve a complete response, and resistance mechanisms are poorly understood. The role of inflammation and immune cell biology in this setting is under-investigated. In this study, we profiled the inflammatory protein secretome of normal (non-cancer) (n = 8) and malignant rectal tissue (n = 12) pre- and post-radiation in human ex vivo explant models and examined the influence of these untreated and treated secretomes on dendritic cell biology (n = 8 for cancer and normal). These resultant profiles were correlated with patient clinical characteristics. Nineteen factors were secreted at significantly higher levels from the rectal cancer secretome when compared to the normal rectal secretome; Flt-1, P1GF, IFN-γ, IL-6, IL-10, CCL20, CCL26, CCL22, CCL3, CCL4, CCL17, GM-CSF, IL-12/IL-23p40, IL-17A, IL-1α, IL-17A/F, IL-1RA, TSLP and CXCL10 (p < 0.05). Radiation was found to have differential effects on normal rectal tissue and rectal cancer tissue with increased IL-15 and CCL22 secretion following radiation from normal rectal tissue explants (p < 0.05), while no significant alterations were observed in the irradiated rectal cancer tissue. Interestingly, however, the irradiated rectal cancer secretome induced the most potent effect on dendritic cell maturation via upregulation of CD80 and PD-L1. Patient's visceral fat area correlated with secreted factors including CCL20, suggesting that obesity status may alter the tumour microenvironment (TME). These results suggest that radiation does not have a negative effect on the ability of the rectal cancer TME to induce an immune response. Understanding these responses may unveil potential therapeutic targets to enhance radiation response and mitigate normal tissue injury. Tumour irradiation in this cohort enhances innate immune responses, which may be harnessed to improve patient treatment outcome.

Keywords: dendritic cells; inflammation; radiotherapy; rectal cancer; tumour immunology.

Mycosis fungoides (MF) is the most common primary cutaneous T-cell lymphoma. While initially restricted to the skin, malignant cells can appear in blood, bone marrow and secondary lymphoid organs in later disease stages. However, only little is known about phenotypic and functional properties of malignant T cells in relationship to tissue environments over the course of disease progression. We thus profiled the tumor micromilieu in skin, blood and lymph node in a patient with advanced MF using single-cell RNA sequencing combined with V-D-J T-cell receptor sequencing. In skin, we identified clonally expanded T-cells with characteristic features of tissue-resident memory T-cells (T_RM, CD69⁺CD27^-NR4A1⁺RGS1⁺AHR⁺ ). In blood and lymph node, the malignant clones displayed a transcriptional program reminiscent of a more central memory-like phenotype (KLF2⁺TCF7⁺S1PR1⁺SELL⁺CCR7⁺ ), while retaining tissue-homing receptors (CLA, CCR10). The skin tumor microenvironment contained potentially tumor-permissive myeloid cells producing regulatory (IDO1) and Th2-associated mediators (CCL13, CCL17, CCL22). Given their expression of PVR, TNFRSF14 and CD80/CD86, they might be under direct control by TIGIT⁺CTLA4⁺CSF2⁺TNFSF14⁺ tumor cells. In sum, this study highlights the adaptive phenotypic and functional plasticity of MF tumor cell clones. Thus, the T_RM-like phenotype enables long-term skin residence of MF cells. Their switch to a T_CM-like phenotype with persistent skin homing molecule expression in the circulation might explain the multi-focal nature of MF.

Keywords: central memory T cells; cutaneous T-cell lymphoma (CTCL); mycosis fungoides; single-cell RNA sequencing; tissue resident memory T cells.

Background and aim: This study evaluates a novel benzylidene-chromanone derivative, FNF-12, for efficacy in in vitro and in vivo asthma models.

Methods: Rat basophilic leukemia (RBL-2H3) and acute monocytic leukemia (THP-1)-derived M2 macrophages were used. Human whole blood-derived neutrophils and basophils were employed. Flow cytometry was used for studying key signalling proteins. Platelet activation factor (PAF)-induced asthma model in guinea pigs was used for in vivo studies.

Results: The chemical structure of FNF-12 was confirmed with proton-nuclear mass resonance (NMR) and mass spectroscopy. FNF-12 controlled degranulation in RBL-2H3 cells with an IC₅₀ value of 123.7 nM and inhibited TNF-α release from these cells in a dose-responsive way. The compound effectively controlled the migration and elastase release in activated neutrophils. IC₅₀ value in the FcεRI-basophil activation assay was found to be 205 nM. FNF-12 controlled the release of lipopolysaccharide (LPS)-induced interleukin-10, I-309/CCL1 and MDC/CCL22 in THP-1 derived M2 macrophages. The compound suppressed LPS-induced mitogen activated protein kinase (MAPK)-p-p38 and nuclear factor kappa B(NF-kB)-p-p65 expression in these cells. A dose-dependent decrease in the accumulation of total leucocytes, eosinophils, neutrophils and macrophages was observed in PAF-induced animal models.

Conclusion: FNF-12 was able to control the inflammatory responses in in vitro and in vivo asthma models, which may be driven by controlling M2-related Th2 cytokines via MAPK and NF-kB signaling.

Keywords: Asthma; Benzylidene-chromanone; MAPK-NF-kB; Platelet activation factor; Th2 cytokines.

New treatments are needed for patients with cutaneous T cell lymphoma (CTCL), particularly for advanced mycosis fungoides (MF) and Sezary syndrome (SS). The immunopathology of MF and SS is complex, but recent advances in tumor microenvironment understanding have identified CCR4 as a promising therapeutic target. CCR4 is widely expressed on malignant T cells and regulatory T cells (Tregs) in the skin and peripheral blood of patients with MF and SS. The interaction of CCR4 with its dominant ligands CCL17 and CCL22 plays a critical role in the development and progression of CTCL, facilitating the movement into, and accumulation of, CCR4-expressing T cells in the skin and recruiting CCR4-expressing Tregs into the tumor microenvironment. Expression of CCR4 is upregulated at all stages of MF and in SS, increasing with advancing disease. Several CCR4-targeted therapies are being evaluated, including 'chemotoxins' targeting CCR4 via CCL17, CCR4-directed chimeric antigen receptor-modified T cell therapies, small-molecule CCR4 antagonists, and anti-CCR4 monoclonal antibodies. Only one is currently approved: mogamulizumab, a defucosylated, fully-humanized anti-CCR4 monoclonal antibody for the treatment of relapsed/refractory MF and SS. Clinical trial data confirm that mogamulizumab is an effective and well-tolerated treatment for relapsed/refractory MF or SS, demonstrating the clinical value of targeting CCR4. This article is protected by copyright. All rights reserved.

Keywords: CCR4; Sezary syndrome; cutaneous T cell lymphoma; mogamulizumab; mycosis fungoides.

The hallmark of preeclampsia (PE) is a shift toward persistent inflammatory response, accompanied by endothelial dysfunction. The driving forces in PE are proinflammatory cytokine and growth factors, in parallel with reduced functionality of anti-inflammatory effectors, like regulatory T cells are observed. Unfortunately, no conclusive mechanism underlying preeclampsia has been identified. For this reason, research on preeclampsia is needed to provide a state of the art understanding of the pathophysiology, identification of new diagnostics tools and the development of targeted therapies. The 68 patients were divided into three groups: gestational hypertension (GH) group (n = 19) and PE group (n = 28) and a control group (n = 21). We have tested a set of 53 cytokines, chemokines and growth factors in preeclampsia and gestational hypertension, and then compared them with normal pregnancies. Using a diagnostic test assessment characteristic parameters (IL-22, MDC/CCL22, IL-2/IL-4 ratio) have been identified and cut-off values have been proposed to diagnose preeclampsia. All parameters had high negative or positive predictive values, above 80%. In conclusion, we have proposed a potential set of immune parameters to diagnose preeclampsia.

Keywords: cytokine; gestational hypertension; growth factors; inflammatory response; preeclampsia.

Immunity plays a key role in epithelial ovarian cancer (EOC) progression with a well-documented correlation between patient survival and high intratumoral CD8+ to T regulatory cell (Treg) ratios. We previously identified dysregulated DPP4 activity in EOCs as a potentially immune-disruptive influence contributing to a reduction in CXCR3-mediated T-cell infiltration in solid tumours. We therefore hypothesized that inhibition of DPP4 activity by sitagliptin, an FDA-approved inhibitor, would improve T-cell infiltration and function in a syngeneic ID8 mouse model of EOC. Daily oral sitagliptin at 50 mg/kg was provided to mice with established primary EOCs. Sitagliptin treatment decreased metastatic tumour burden and significantly increased overall survival and was associated with significant changes to the immune landscape. Sitagliptin increased overall CXCR3-mediated CD8+ T-cell trafficking to the tumour and enhanced the activation and proliferation of CD8+ T-cells in tumour tissue and the peritoneal cavity. Substantial reductions in suppressive cytokines, including CCL2, CCL17, CCL22 and IL-10, were also noted and were associated with reduced CD4+ CD25+ Foxp3+ Treg recruitment in the tumour. Combination therapy with paclitaxel, however, typical of standard-of-care for patients in palliative care, abolished CXCR3-specific T-cell recruitment stimulated by sitagliptin. Our data suggest that sitagliptin may be suitable as an adjunct therapy for patients between chemotherapy cycles as a novel approach to enhance immunity, optimise T-cell-mediated function and improve overall survival.

Keywords: DPP4; ID8; T-cell; immune; ovarian cancer; sitagliptin; syngeneic.

Bacteriophages (phages) are viruses of bacteria. Despite the growing progress in research on phage interactions with eukaryotic cells, our understanding of the roles of phages and their potential implications remains incomplete. The objective of this study was to investigate the effects of the Staphylococcus aureus phage vB_SauM_JS25 on murine norovirus (MNV) replication. Experiments were performed using the RAW 264.7 cell line. After phage treatment, MNV multiplication was significantly inhibited, as indicated by real-time quantitative polymerase chain reaction (RT-qPCR) analysis, western blotting, the 50% tissue culture infectious dose and immunofluorescence. Furthermore, we revealed transcriptional changes in phage/MNV co-incubated RAW 264.7 cells through RNA sequencing (RNA-seq) and bioinformatic analysis. Our subsequent analyses revealed that the innate immune response might play an important role in restriction of MNV replication, such as the cellular response to IFN-γ and response to IFN-γ. Additionally, gene expression of IL-10, Arg-1, Ccl22, GBP2, GBP3, GBP5, and GBP7 was increased significantly, which indicated a strong correlation between RT-qPCR and RNA-seq results. Furthermore, phage treatment activated guanylate binding proteins (GBPs), as revealed by RT-qPCR analysis, western blotting, and confocal microscopy. Taken together, these data suggest that the phage affects the innate response, such as the IFN-inducible GTPases and GBPs, and therefore exerts an antiviral effect in vitro. Collectively, our findings provide insights into the interactions of immune cells and phages, which establish phage-based antiviral effects.

Keywords: antiviral agents; bacteriophages; interferons; macrophages; norovirus.

CCR4 is a chemokine receptor mainly expressed by T cells. It is the receptor for two CC chemokine ligands, CCL17 and CCL22. Originally, the expression of CCR4 was described as highly selective for helper T type 2 (Th2) cells. Later, its expression was extended to other T cell subsets such as regulatory T (Treg) cells and Th17 cells. CCR4 has long been regarded as a potential therapeutic target for allergic diseases such as atopic dermatitis and bronchial asthma. Furthermore, the findings showing that CCR4 is strongly expressed by T cell malignancies such as adult T cell leukemia/lymphoma (ATLL) and cutaneous T cell lymphomas (CTCLs) have led to the development and clinical application of the fully humanized and glyco-engineered monoclonal anti-CCR4 Mogamulizumab in refractory/relapsed ATLL and CTCLs with remarkable successes. However, Mogamulizumab often induces severe adverse events in the skin possibly because of its efficient depletion of Treg cells. In particular, treatment with Mogamulizumab prior to allogenic hematopoietic stem cell transplantation (allo-HSCT), the only curative option of these T cell malignancies, often leads to severe glucocorticoid-refractory graft-versus-host diseases. The efficient depletion of Treg cells by Mogamulizumab has also led to its clinical trials in advanced solid tumors singly or in combination with immune checkpoint inhibitors. The main focus of this review is CCR4; its expression on normal and malignant T cells and its significance as a therapeutic target in cancer immunotherapy.

Keywords: ATLL; CCR4; CTLL; HAM/TSP; HTLV-1; Mogamulizumab; T cell subset; chemokine; chemokine receptor; immune check point inhibitor.

Abnormality in the number and function of CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Tregs) in peripheral blood has been linked to the initiation and progression of rheumatoid arthritis (RA). Effect of chemokine CCL22 on the number of Tregs in CD4⁺ T cells and the underlying mechanism were investigated. Downregulation of peripheral Tregs were observed while upregulation of serum chemokine CCL22 in RA patients. Tregs count and the expression of FOXP3 (Tregs function-related maker) and phosphorylated-signal transducer and activator of transcription 5 (p-STAT5) in CD4⁺ T cells from RA patients were increased while C-C chemokine receptor 4 (CCR4) was decreased by anti-CCL22 antibody, however, recombinant CCL22 resulted in the opposite effects in CD4⁺ T cells from the healthy control. STAT5 inhibitor significantly reversed the effects of anti-CCL22 antibody. Similarly, sinomenine, an anti-arthritis drug, which decreased CCL22 and CCR4, showed the same trends as the above events, and was reversed by recombinant CCL22 or STAT5 inhibitor. Collectively, anti-CCL22 induced the number of Tregs via STAT5 pathway, leading to expansion of Tregs and subsequently to control of the autoimmune reaction in RA patients. Our study provides s novel strategy for RA treatment.