Chimeric Antigen Receptor-Redirected Regulatory T Cells Suppress Experimental Allergic Airway Inflammation, a Model of Asthma

Cellular therapy with chimeric antigen receptor (CAR)-redirected cytotoxic T cells has shown impressive efficacy in the treatment of hematologic malignancies. We explored a regulatory T cell (Treg)-based therapy in the treatment of allergic airway inflammation, a model for asthma, which is characterized by an airway hyper-reactivity (AHR) and a chronic, T helper-2 (Th2) cell-dominated immune response to allergen. To restore the immune balance in the lung, we redirected Tregs by a CAR toward lung epithelia in mice upon experimentally induced allergic asthma, closely mimicking the clinical situation. Adoptively transferred CAR Tregs accumulated in the lung and in tracheobronchial lymph nodes, reduced AHR and diminished eosinophilic airway inflammation, indicated by lower cell numbers in the bronchoalveolar lavage fluid and decreased cell infiltrates in the lung. CAR Treg cells furthermore prevented excessive pulmonary mucus production as well as increase in allergen-specific IgE and Th2 cytokine levels in exposed animals. CAR Tregs were more efficient in controlling asthma than non-modified Tregs, indicating the pivotal role of specific Treg cell activation in the affected organ. Data demonstrate that lung targeting CAR Treg cells ameliorate key features of experimental airway inflammation, paving the way for cell therapy of severe allergic asthma

Absence of Regulatory T Cells Causes Phenotypic and Functional Switch in Murine Peritoneal Macrophages

Tissue macrophages are important components of tissue homeostasis and inflammatory pathologies. In the peritoneal cavity, resident macrophages interact with a variety of immune cells and can exhibit broad range of phenotypes and functions. Forkhead-box-P3 (FOXP3)+ regulatory T cells (Tregs) play an indispensable role in maintaining immunological tolerance, yet whether, and how the pathological condition that results from the lack of functional Tregs affects peritoneal macrophages (PM) is largely unknown. We used FOXP3-deficient scurfy (Sf) mice to investigate PM behavior in terms of the missing crosstalk with Tregs. Here, we report that Treg deficiency induced a marked increase in PM numbers, which was reversed after adoptive transfer of CD4+ T cells or neutralization of macrophage colony-stimulating factor. Ex vivo assays demonstrated a pro-inflammatory state of PM from Sf mice and signs of excessive activation and exhaustion. In-depth immunophenotyping of Sf PM using single-cell chipcytometry and transcriptome analysis revealed upregulation of molecules involved in the initiation of innate and adaptive immune responses. Moreover, upon transfer to non-inflammatory environment or after injection of CD4+ T cells, PM from Sf mice reprogramed their functional phenotype, indicating remarkable plasticity. Interestingly, frequencies, and immune polarization of large and small PM subsets were dramatically changed in the FOXP3-deficient mice, suggesting distinct origin and specialized function of these subsets in inflammatory conditions. Our findings demonstrate the significant impact of Tregs in shaping PM identity and dynamics. A better understanding of PM function in the Sf mouse model may have clinical implication for the treatment of immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, and other forms of immune-mediated enteropathies

Chimeric Antigen Receptor-Redirected Regulatory T Cells Suppress Experimental Allergic Airway Inflammation, a Model of Asthma

Cellular therapy with chimeric antigen receptor (CAR)-redirected cytotoxic T cells has shown impressive efficacy in the treatment of hematologic malignancies. We explored a regulatory T cell (Treg)-based therapy in the treatment of allergic airway inflammation, a model for asthma, which is characterized by an airway hyper-reactivity (AHR) and a chronic, T helper-2 (Th2) cell-dominated immune response to allergen. To restore the immune balance in the lung, we redirected Tregs by a CAR toward lung epithelia in mice upon experimentally induced allergic asthma, closely mimicking the clinical situation. Adoptively transferred CAR Tregs accumulated in the lung and in tracheobronchial lymph nodes, reduced AHR and diminished eosinophilic airway inflammation, indicated by lower cell numbers in the bronchoalveolar lavage fluid and decreased cell infiltrates in the lung. CAR Treg cells furthermore prevented excessive pulmonary mucus production as well as increase in allergen-specific IgE and Th2 cytokine levels in exposed animals. CAR Tregs were more efficient in controlling asthma than non-modified Tregs, indicating the pivotal role of specific Treg cell activation in the affected organ. Data demonstrate that lung targeting CAR Treg cells ameliorate key features of experimental airway inflammation, paving the way for cell therapy of severe allergic asthma

Decreased production of class-switched antibodies in neonatal B cells is associated with increased expression of miR-181b

The increased susceptibility to infections of neonates is caused by an immaturity of the immune system as a result of both qualitative and quantitative differences between neonatal and adult immune cells. With respect to B cells, neonatal antibody responses are known to be decreased. Accountable for this is an altered composition of the neonatal B cell compartment towards more immature B cells. However, it remains unclear whether the functionality of individual neonatal B cell subsets is altered as well. In the current study we therefore compared phenotypical and functional characteristics of corresponding neonatal and adult B cell subpopulations. No phenotypic differences could be identified with the exception of higher IgM expression in neonatal B cells. Functional analysis revealed differences in proliferation, survival, and B cell receptor signaling. Most importantly, neonatal B cells showed severely impaired class-switch recombination (CSR) to IgG and IgA. This was associated with increased expression of miR-181b in neonatal B cells. Deficiency of miR-181b resulted in increased CSR. With this, our results highlight intrinsic differences that contribute to weaker B cell antibody responses in newborns

B cells control maternofetal priming of allergy and tolerance in a murine model of allergic airway inflammation

Background: Allergic asthma is a chronic lung disease resulting from inappropriate immune responses to environmental antigens. Early tolerance induction is an attractive approach for primary prevention of asthma. Objective: We analyzed the mechanisms of perinatal tolerance induction to allergens, with particular focus on the role of B cells in preconception and early intrauterine immune priming. Methods: Wild-type (WT) and B cell-deficient mice received ovalbumin (OVA) intranasally before mating. Their offspring were analyzed in a murine model of allergic airway inflammation. Results: Although antigen application before conception protected WT progeny from allergy, it aggravated allergic airway inflammation in B cell-deficient offspring. B-cell transfer restored protection, demonstrating the crucial role of B cells in perinatal tolerance induction. Effective diaplacentar allergen transfer was detectable in pregnant WT mice but not in pregnant B-cell knockout dams, and a ntigen concentrations in WT amniotic fluid (AF) were higher than in IgG-free AF of B cell-deficient dams. Application of OVA/IgG immune complexes during pregnancy boosted OVA uptake by fetal dendritic cells (DCs). Fetal DCs in human subjects and mice expressed strikingly higher levels of Fcγ receptors compared with DCs from adults and were highly efficient in taking up OVA/IgG immune complexes. Moreover, murine fetal DCs effectively primed antigen-specific forkhead box P3+ regulatory T cells after in vitro coincubation with OVA/IgG-containing AF. Conclusion: Our data support a decisive role for B cells and immunoglobulins during in utero tolerance priming. These findings improve the understanding of perinatal immunity and might support the development of effective primary prevention strategies for allergy and asthma in the future

MitoNEET is increased by 2-AAPA.

<p>(A) Addition of GSR-inhibitor 2-AAPA (10 μM) led to a mild increase of mitoNEET-mRNA (n = 5) (B) while protein was increased 4-fold (n = 6) after H/R. (C) Representative Western Blots is presented. Data are expressed as % of H/R control. Real-time RT-PCR signals were normalized to hypoxanthine phosphoribosyl-transferase (HPRT) gene expression and data are expressed as 2^-ΔΔCT.</p

MitoNEET plays a role for oxidative stress induced apoptosis in cardiac HL-1 cells.

<p>HL-1 cells were transfected with silencing RNA (siRNA) directed against mitoNEET and non-specific-siRNA as control and subjected to 3h hypoxia followed by 1h of reoxygenation (H/R). (A) Densitometric analysis of Western Blots revealed aggravated activation of caspase-3 in mitoNEET-knockdown (mitoN-KD) cells after H/R compared to hypoxic controls (n = 11). (B) MitoNEET-KD showed no effect on lactate dehydrogenase (LDH) release after H/R measured in culture supernatants (n = 10). LDH was quantified as U/L by a routine clinical analyzer and is expressed as % of H/R control. (C,D) Overexpression of mitoNEET in HL-1 cells caused a significant decrease in apoptosis in mitoNEET overexpressing cells compared to H/R control cells. Expression of mitoNEET (n = 5) and cleaved caspase (n = 7) is shown in representative Western Blots and was densitometrically measured as % of H/R control. (E-G) H/R-induced apoptosis in control- and mitoNEET-KD cells was reduced by two different antioxidants, superoxide scavenger Tiron (10 mM, n = 5) and esterified glutathione compound GSH-MEE (glutathione reduced ethyl ester, 2 mM, n = 6) as demonstrated by representative Western Blots (F-G). Data were analyzed densitometrically, normalized to housekeeping gene expression and are expressed as % of H/R control.</p

Chemical inhibition of glutathione reductase (GSR) reduces oxidative stress-induced apoptosis.

<p>(A) Chemical GSR-inhibitor 2-AAPA (10 μM) reduced GSR-activity to 20% (n = 5). GSR-activity was determined by a colorimetric glutathione reductase assay. Thereby GSR reduces GSSG to GSH that reacts with 5, 5′-Dithiobis (2-nitrobenzoic acid) (DTNB) to generate yellow TNB^2-, which was measured at 405 nm in a microplate reader. GSR-activity was calculated, normalized to protein amount (mU/mg protein) and is expressed as % of H/R control. (B) Blockade of GSR-activity by 10 μM 2-AAPA decreased levels of activated caspase-3 in HL-1 cells after H/R (n = 5). (C) Representative Western Blot image is shown. Data were densitometrically analyzed and are expressed as % of H/R control. (D) TUNEL staining was utilized to determine apoptosis in HL-1 cells by the use of MEBSTAIN Apoptosis Kit II according to manufacturer`s protocol (MBL, Woburn, MA, USA). Data are presented as TUNEL-positive nuclei per total nuclei (n = 5).</p

2-AAPA effect on mitoNEET is abolished by addition of glutathione compound GSH-MEE.

<p>MitoNEET-protein amount of HL-1 cells was clearly increased after addition of GSR-inhibitor 2-AAPA (10 μM) determined after H/R by Western Blot. This effect was completely reversed by additon of glutathione compound GSH-MEE (2 mM, n = 4–5) as demonstrated by a representative Western Blot. Data are expressed as % of H/R control.</p

MitoNEET Protects HL-1 Cardiomyocytes from Oxidative Stress Mediated Apoptosis in an <i>In Vitro</i> Model of Hypoxia and Reoxygenation

<div><p>The iron-sulfur cluster containing protein mitoNEET is known to modulate the oxidative capacity of cardiac mitochondria but its function during myocardial reperfusion injury after transient ischemia is unknown. The purpose of this study was to analyze the impact of mitoNEET on oxidative stress induced cell death and its relation to the glutathione-redox system in cardiomyocytes in an <i>in vitro</i> model of hypoxia and reoxygenation (H/R). Our results show that siRNA knockdown (KD) of mitoNEET caused an 1.9-fold increase in H/R induced apoptosis compared to H/R control while overexpression of mitoNEET caused a 53% decrease in apoptosis. Necrosis was not affected. Apoptosis of both, mitoNEET-KD and control cells was diminished to comparable levels by using the antioxidants Tiron and glutathione compound glutathione reduced ethyl ester (GSH-MEE), indicating that mitoNEET-dependent apoptosis is mediated by oxidative stress. The interplay between mitoNEET and glutathione redox system was assessed by treating cardiomyocytes with 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthio-carbonylamino) phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA), known to effectively inhibit glutathione reductase (GSR) and to decrease the GSH/GSSG ratio. Surprisingly, inhibition of GSR-activity to 20% by 2-AAPA decreased apoptosis of control and mitoNEET-KD cells to 23% and 25% respectively, while at the same time mitoNEET-protein was increased 4-fold. This effect on mitoNEET-protein was not accessible by mitoNEET-KD but was reversed by GSH-MEE. In conclusion we show that mitoNEET protects cardiomyocytes from oxidative stress-induced apoptosis during H/R. Inhibition of GSH-recycling, GSR-activity by 2-AAPA increased mitoNEET-protein, accompanied by reduced apoptosis. Addition of GSH reversed these effects suggesting that mitoNEET can in part compensate for imbalances in the antioxidative glutathione-system and therefore could serve as a potential therapeutic approach for the oxidatively stressed myocardium.</p></div