The increased number of epithelial mast cells in nasal polyps and adjacent turbinates is not allergy-dependent

Respiratory epithelial mast cells are an expression of airway inflammatory processes. Nasal epithelial mast cells are known to be increased in allergic rhinitis and have now been examined in patients with nasal polyps. Metachromatic cell counts (mean +/- standard error) expressed as the sum of large mast cells, atypical mast cells and basophils in epithelial scrapings of the inferior turbinates, assessed after Carnoy's fixation and toluidine blue staining (pH 0.5), were 37.5 +/- 29 in non-allergic normal control subjects (n = 11), 435 +/- 130 in polyp patients who were allergic (n = 18), and 699 +/- 267 in polyp patients who were not allergic (n = 8). Metachromatic cell counts in epithelial scrapings obtained in vivo from nasal polyps of allergic patients (n = 8) were 1769 +/- 962, and 2308 +/- 1544 from polyps of non-allergic patients (n = 5); metachromatic counts were 2089 +/- 633 in epithelial scrapings from excised polyps of allergic patients (n = 14) and 2214 +/- 640 from polyps of non-allergic patients (n = 13). It is concluded that the number of metachromatic cells in the epithelium of nasal polyps and the adjacent nasal mucosa is elevated compared with normal nasal epithelium and the increased number does not depend upon allergy.     

Heme oxygenase-1 is not required for mouse regulatory T cell development and function

CD4 regulatory T cells (Treg) ensure peripheral tolerance to self-antigens and limit the deleterious effects associated with inflammatory and immune responses by mechanisms that remain to be fully understood. The enzyme heme oxygenase-1 (HO-1), through its known anti-inflammatory activity, is a candidate for a functional role in Treg activity. We compared wild-type and heme oxygenase-1-deficient (hmox-1(-/-)) mice in order to assess the role of HO-1 in mouse Treg development and function under physiologic conditions. The frequency of CD25+ and Foxp3+ Treg was similar in hmox-1(-/-) and hmox-1(+/+) mice. More importantly, CD4+ CD25+ Treg purified from either hmox-1(-/-) or hmox-1(+/+) mice were equally efficient in controlling the proliferation in vitro and the expansion in vivo of CD4+ CD25- T cells, whether or not these responder cells expressed HO-1. In addition, induction of expression of HO-1 in vivo did not affect Treg suppressor function. As shown before, expression of HO-1 was higher in Treg than in naive T cells; however, naturally activated Foxp3- T cells displayed equal amount of HO-1 mRNA as Treg. Finally, we conclude that under physiological conditions in mice, Treg development, maintenance and function are independent of HO-1 activity.     

Harnessing proteases for T regulatory cell immunotherapy

The immune system is tightly regulated by a subset of T cells defined as regulatory T cells (Tregs). Tregs maintain immune homeostasis by restraining unwarranted immune cell activation and effector function. Here, we discuss an important but underappreciated role of proteases in controlling Treg function. Proteases regulate a number of vital processes that determine T cell immune responses and some of them such as furin, ADAM (through regulating LAG receptor), MALT, and asparaginyl endopeptidase are implicated in Treg immunobiology. Targeted protease inhibition, using either small molecule inhibitors or gene deficient mice has demonstrated their specificity in modulating Treg function in experimental murine models. These data further highlight the ability of proteases to specifically regulate Tregs but no other T effector lineages. Taken together, it is apparent that incorporating proteases as targets within Treg cell engineering protocols may enable generation of robust Treg cellular therapeutics. These engineered Tregs may possess enhanced regulatory function along with resistance to lineage deviation in inflammatory disease such as colitis and graft versus host disease. Within this review, we summarize research on the role of proteases in regulating Treg function and discuss the translational potential of harnessing Treg function by targeting protease driven regulatory pathways.     

P21‐activated kinase 2 is essential in maintenance of peripheral Foxp3+ regulatory T cells

The p21‐activated kinase 2 (Pak2), an effector molecule of the Rho family GTPases Rac and Cdc42, regulates diverse functions of T cells. Previously, we showed that Pak2 is required for development and maturation of T cells in the thymus, including thymus‐derived regulatory T (Treg) cells. However, whether Pak2 is required for the functions of various subsets of peripheral T cells, such as naive CD4 and helper T‐cell subsets including Foxp3⁺ Treg cells, is unknown. To determine the role of Pak2 in CD4 T cells in the periphery, we generated inducible Pak2 knockout (KO) mice, in which Pak2 was deleted in CD4 T cells acutely by administration of tamoxifen. Temporal deletion of Pak2 greatly reduced the number of Foxp3⁺ Treg cells, while minimally affecting the homeostasis of naive CD4 T cells. Pak2 was required for proliferation and Foxp3 expression of Foxp3⁺ Treg cells upon T‐cell receptor and interleukin‐2 stimulation, differentiation of in vitro induced Treg cells, and activation of naive CD4 T cells. Together, Pak2 is essential in maintaining the peripheral Treg cell pool by providing proliferation and maintenance signals to Foxp3⁺ Treg cells.     

Ratio of myeloid and plasmacytoid dendritic cells and TH2 skew in CRS with nasal polyps

Background: The role of myeloid and plasmacytoid dendritic cells and its consequences for the T_H2 skew in chronic rhinosinusitis (CRS) with nasal polyps (CRSNP⁺) should be detailed. Methods: In 18 CRS patients without nasal polyps (CRSNP^?), 35 CRSNP⁺ patients and 22 patients with nasal structural abnormalities without rhinosinusitis (controls), dendritic cells (DC) were differentiated into myeloid (mDC) and plasmacytoid (pDC) subtypes using an antibody cocktail including CD1c (BDCA‐1) and CD303 (BDCA‐2) in peripheral blood mononuclear cells (PBMC) and single cell preparations of sinonasal mucosa by flow cytometry. Moreover, cells were analysed for expression of CD45, CD3, CD4, CXCR3 (T_H1) and CCR4 (T_H2) and IFN‐γ, IL‐5, TGF‐β1, TGF‐β2, ECP and total IgE in nasal secretions were determined. As a possible confounder, Staphylococcus aureus in nasal lavages was detected. Results: The tissue mDC/pDC‐ratio was 1.7 (1.0–2.4) in controls, 3.0 (1.8–4.0) in CRSNP^? and 0.8 (0.6–1.0) in CRSNP⁺ (P < 0.01). In tissue samples, the T_H1/T_H2 ratio was 12.6 (6.4–16.0) in controls, 12.5 (6.9–21.2) in CRSNP^? and 1.8 (1.3–3.6) in CRSNP⁺ (median and interquartile range, P < 0.001). Less pronounced differences were found in PBMC. S. aureus detection rates or TGF‐β levels did not differ between patient groups and S. aureus detection had no influence on the parameters investigated. Conclusion: A significant T_H2 skew in CRSNP⁺ could be confirmed on the cellular level. It was driven by low myeloid dendritic cell numbers. The T_H2 skew did not correlate with S. aureus detection. The data support the concept that CRSNP⁺ and CRSNP^? are pathophysiologically distinct.     

Corticosteroid therapy increases membrane-tethered while decreases secreted mucin expression in nasal polyps

Background: Mucus hypersecretion is a hallmark of nasal polyposis (NP). Corticosteroids (CS) are first‐line treatment for NP, decreasing their size and inflammatory component. However, their effect on mucin production is not well‐understood. The aim of this (pilot) study was to investigate CS effect on mucin expression in NP. Methods: Patients were randomized in control (n = 9) and treatment (oral prednisone for 2 weeks and intranasal budesonide for 12 weeks; n = 23) groups. Nasal polyposis from nonasthmatic (NP; n = 13), aspirin‐tolerant (NP‐ATA; n = 11) and aspirin‐intolerant (NP‐AIA; n = 8) asthmatics were studied. Nasal polyposis biopsies were obtained before (w0) and after 2 (w2) and 12 (w12) weeks of CS treatment. Secreted (MUC5AC, MUC5B and MUC8) and membrane‐tethered (MUC1, MUC4) mucins (immunohistochemistry) and goblet cells (Alcian blue‐periodic acid Schiff) were quantified in both epithelium and glands. Rhinorrea and nasal obstruction were also assessed. Results: At w2, steroids increased MUC1 (from 70 to 97.5) and MUC4 (from 80 to 100) in NP‐ATA patients’ epithelium compared with baseline (w0). At w12, steroids decreased MUC5AC (from 40 to 5) and MUC5B (from 45 to 2.5) in NP‐ATA patients’ epithelium and glands, respectively, compared with baseline. No mucin presented significant changes in NP‐AIA patients. MUC5AC and MUC5B expression correlated with goblet and mucous cell numbers, respectively, and MUC5AC also with rhinorrea score. Conclusions: These results suggest: (i) CS up‐regulate membrane (MUC1, MUC4) while down‐regulate secreted (MUC5AC, MUC5B) mucins; (ii) there exists a link between secreted mucin expression and goblet cell hyperplasia; and (iii) NP from AIA may develop resistance to CS treatment.     

The expression of dendritic cell subsets in severe chronic rhinosinusitis with nasal polyps is altered

Background

Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized as a Th2-driven disease.Activated dendritic cells (DCs) are the main T-cell activators; their role in the chronic inflammatory process of nasal polyposis is still unclear.

Methods

The regulation of DC subsets was analyzed in nasal polyp tissue from CRSwNP patients and compared to inferior turbinate tissue from healthy subjects. Tissue localization and expression of both plasmacytoid and myeloid DCs were assayed by means of immunohistochemistry and flow cytometry. Plasmacytoid DCs were also assayed by PCR, and tissue homogenates were assayed for various inflammatory markers.

Results

The number of plasmacytoid (pDCs) and myeloid (mDCs) dendritic cells was significantly increased in nasal polyp tissue when compared to non-inflamed nasal mucosa. The number of pDCs, but not mDCs, was down-regulated in more severe cases (nasal polyps with asthma) and varied with the cytokine milieu. The amount of pDCs was significantly decreased in IL5+IFNγ – nasal polyp tissue compared to tissues with high IFNγ levels (IL5+IFNγ+). Furthermore, levels of indoleamine 2,3-dioxygenase were increased in nasal polyp compared to inferior turbinate tissue and correlated negatively with the number of pDCs.

Conclusions

There is an altered balance of pDC and mDC numbers in nasal polyp tissue. pDCs seem to be more susceptible to an inflammatory cytokine milieu and may play a crucial role in disease severity.     

Effects of in vivo injection of anti-chicken CD25 monoclonal antibody on regulatory T cell depletion and CD4+CD25- T cell properties in chickens

Regulatory T cells (Tregs) are defined as CD4⁺CD25⁺ cells in chickens. This study examined the effects of an anti-chicken CD25 monoclonal antibody injection (0.5 mg/bird) on in vivo depletion of Tregs and the properties of CD4⁺CD25⁻ cells in Treg-depleted birds. The CD4⁺CD25⁺ cell percentage in the blood was lower at 8 d post injection than at 0 d. Anti-CD25-mediated CD4⁺CD25⁺ cell depletion in blood was maximum at 12 d post injection. The anti-CD25 antibody injection depleted CD4⁺CD25⁺ cells in the spleen and cecal tonsils, but not in the thymus, at 12 d post antibody injection. CD4⁺CD25⁻ cells from the spleen and cecal tonsils of birds injected with the anti-chicken CD25 antibody had higher proliferation and higher IL-2 and IFNγ mRNA amounts than the controls at 12 d post injection. At 20 d post injection, CD4⁺CD25⁺ cell percentages in the blood, spleen and thymus were comparable to that of the 0 d post injection. It could be concluded that anti-chicken CD25 injection temporarily depleted Treg population and increased and IL-2 and IFNγ mRNA amounts in CD4⁺CD25⁻ cells at 12 d post injection.     

In‐vitro effect of pembrolizumab on different T regulatory cell subsets

Programmed death‐1 (PD‐1) and interactions with PD‐ligand 1 (PD‐L1) play critical roles in the tumour evasion of immune responses through different mechanisms, including inhibition of effector T cell proliferation, reducing cytotoxic activity, induction of apoptosis in tumour‐infiltrating T cells and regulatory T cell (T_reg) expansion. Effective blockade of immune checkpoints can therefore potentially eliminate these detrimental effects. The aim of this study was to investigate the effect of anti‐PD‐1 antibody, pembrolizumab, on various T_reg subpopulations. Peripheral blood mononuclear cells (PBMC) from healthy donors (HD) and primary breast cancer patients (PBC) were treated in vitro with pembrolizumab, which effectively reduced PD‐1 expression in both cohorts. We found that PD‐1 was expressed mainly on CD4⁺CD25⁺ T cells and pembrolizumab had a greater effect on PD‐1 expression in CD4⁺CD25^? T cells, compared to CD4⁺CD25⁺ cells. In addition, pembrolizumab did not affect the expression levels of T_reg‐related markers, including cytotoxic T lymphocyte antigen‐4 (CTLA‐4), CD15s, latency‐associated peptide (LAP) and Ki‐67. Moreover, we report that CD15s is expressed mainly on forkhead box P3 (FoxP3)^?Helios⁺ T_reg in HD, but it is expressed on FoxP3⁺Helios^? T_reg subset in addition to FoxP3^?Helios⁺ T_reg in PBC. Pembrolizumab did not affect the levels of FoxP3^+/?Helios^+/? T_reg subsets in both cohorts. Taken together, our study suggests that pembrolizumab does not affect T_reg or change their phenotype or function but rather blocks signalling via the PD‐1/PD‐L1 axis in activated T cells.     

Regulation of regulatory T cells in cancer

The inflammatory response to transformed cells forms the cornerstone of natural or therapeutically induced protective immunity to cancer. Regulatory T (Treg) cells are known for their critical role in suppressing inflammation, and therefore can antagonize effective anti-cancer immune responses. As such, Treg cells can play detrimental roles in tumour progression and in the response to both conventional and immune-based cancer therapies. Recent advances in our understanding of Treg cells reveal complex niche-specific regulatory programmes and functions, which are likely to extrapolate to cancer. The regulation of Treg cells is reliant on upstream cues from haematopoietic and non-immune cells, which dictates their genetic, epigenetic and downstream functional programmes. In this review we will discuss how Treg cells are themselves regulated in normal and transformed tissues, and the implications of this cross talk on tumour growth.