Christian Hennig

Allergy prevention starts before conception: Maternofetal transfer of tolerance protects against the development of asthma

BACKGROUND Allergy prevention must start early because environmental exposures during pregnancy and young life determine disease risk. OBJECTIVE In this study we analyzed whether prevention can start even earlier before conception by transfer of immunologic tolerance from the mother to the offspring preventing the offspring from having asthma. METHODS BALB/c mice were orally tolerized with ovalbumin before conception by means of oral application of antigen. The offspring of tolerized and naive mothers were immunized with ovalbumin at 6 weeks and 4 months of age and analyzed in our murine asthma model. RESULTS Although the offspring of naive mothers had an asthma-like phenotype, the offspring of tolerized mice were completely protected, even when immunized as late as 8 months after birth. Critically involved in the tolerance transfer was allergen-specific IgG, levels of which were increased in the sera of the mother, fetus, and pup and breast milk. FcRn(-/-) mice, which cannot transport IgG through the placenta, transferred tolerance to the offspring only when the missing diaplacental IgG transfer was compensated by IgG transfer through breast milk from tolerant mothers but not when weaned by naive wet nurses. Inhibition of IFN-gamma, produced by memory T cells in the offspring, abrogated the protective effect of maternal tolerance, demonstrating a crucial role for IFN-gamma in the maintenance of allergen-specific tolerance. CONCLUSION Our data show that maternal immunologic memory has a significant and persistent effect on the immune response of the offspring.

A versatile platform for comprehensive chip‐based explorative cytometry

Analysis of the immense complexity of the immune system is increasingly hampered by technical limitations of current methodologies, especially for multiparameter‐ and functional analysis of samples containing small numbers of cells. We here present a method, which is based on the stepwise functional manipulation and analysis of living immune cells that are self‐immobilized within microfluidic chips using automated epifluorescence microscopy overcoming current limitations for comprehensive immunophenotyping. Crossvalidation with flow cytometry revealed a 10‐fold increased sensitivity and a comparable specificity. By using small sample volumes and cell numbers (2–10 μl, down to 20,000 cells), we were able to analyze a virtually unlimited number of intracellular and surface markers even on living immune cells. We exemplify the scientific and diagnostic potential of this method by (1) identification and phenotyping of rare cells, (2) comprehensive analysis of very limited sample volume, and (3) deep immunophenotyping of human B‐cells after in vitro differentiation. Finally, we propose an informatic model for annotation and comparison of cytometric data by using an ontology‐based approach. The chip‐based cytometry introduced here turned out to be a very useful tool to enable a stepwise exploration of precious, small cell‐containing samples with an virtually unlimited number of surface‐ and intracellular markers.

ICOS Mediates the Generation and Function of CD4+CD25+Foxp3+ Regulatory T Cells Conveying Respiratory Tolerance

Costimulatory molecules like ICOS are crucial in mediating T cell differentiation and function after allergen contact and thereby strongly affect the immunologic decision between tolerance or allergy development. In this study, we show in two independent approaches that interruption of the ICOS signaling pathway by application of a blocking anti-ICOSL mAb in wild-type (WT) mice and in ICOS−/− mice inhibited respiratory tolerance development leading to eosinophilic airway inflammation, mucus hypersecretion, and Th2 cytokine production in response to OVA sensitization. Respiratory Ag application almost doubled the number of CD4+Foxp3+ regulatory T cells (Tregs) in the lung of WT mice with 77% of lung-derived Tregs expressing ICOS. In contrast, in ICOS−/− mice the number of CD4+CD25+Foxp3+ Tregs did not increase after respiratory Ag application, and ICOS−/− Tregs produced significantly lower amounts of IL-10 than those of WT Tregs. Most importantly, in contrast to WT Tregs, ICOS−/− Tregs did not convey protection when transferred to “asthmatic” recipients demonstrating a strongly impaired Treg function in the absence of ICOS signaling. Our findings demonstrate a crucial role of ICOS for the generation and suppressive function of Tregs conveying respiratory tolerance and support the importance of ICOS as a target for primary prevention strategies.

Immunophenotyping of Cerebrospinal Fluid Cells in Multiple Sclerosis In Search of Biomarkers

IMPORTANCE Cerebrospinal fluid (CSF) is the compartment in closest proximity to the central nervous system (CNS) parenchyma and might reflect immune pathology in inflammatory CNS disorders like multiple sclerosis (MS). Multiparameter flow cytometry is used to characterize immunological alterations in the CSF of patients with MS. OBJECTIVES To present a comprehensive review of the cellular alterations in CSF that distinguish MS from physiological conditions and other CNS disorders; integrate relevant findings into a model of leukocyte trafficking in the CNS; highlight treatment-related changes in leukocyte subsets; and evaluate the potential of CSF immunophenotyping in the search of novel biomarkers in MS. EVIDENCE REVIEW We searched MEDLINE articles published between 1980 and 2013 that include the flow cytometric characterization of leukocyte subsets in the CSF of patients with MS. FINDINGS All of the articles have shown CSF pleocytosis in MS. Interesting results include CSF enrichment of helper T cells (subtypes TH1 and TH17) and regulatory T cells, as well as intrathecal B-cell differentiation resulting in the generation of antibody-producing plasmablasts and plasma cells. Other leukocyte populations, including natural killer cells, monocytes, and dendritic cells, show alterations as well. Characterization of CSF cells increases our understanding of MS pathogenesis and may provide useful biomarkers for individual prognosis and treatment decisions. However, validation in controlled settings is lacking in most cases. CONCLUSIONS AND RELEVANCE With the advent of more sophisticated approaches, immunophenotyping of CSF cells in MS might become increasingly important to correlate cellular subsets with different stages of disease activity and remission. An assessment of CSF cell numbers and composition should be incorporated into clinical trials.

Lung function and inflammation during murine Pseudomonas aeruginosa airway infection

BACKGROUND Following any acute irritation lung function declines rapidly. Reasons for pulmonary deterioration in humans had been attributed to the action of either interleukin-6 or interleukin-8 in the lungs. OBJECTIVES The present study investigates the association between immune response and decline in lung function in a murine bacterial lung infection model. METHODS Upon intratracheal inoculation of C57BL/6J mice with a sublethal dose of Pseudomonas aeruginosa lung function, cytokine, chemokine and cytometry in bronchoalveolar lavage fluid, bacterial counts and lung histology was assessed at 2, 4, 6, 8, 10, 12, 18, 24, 48, 72, 96 and 120 h post inoculation. RESULTS Lung function measured by non-invasive head-out spirometry decreased most strongly between 6 and 10 h post inoculation and required up to 72 h to recover for selected parameters. CFU counts in the lungs peaked at 4h post inoculation with subsequent decline until at 24-48 h post inoculation background levels were reached. Cytokine and chemokine responses could be separated into an early pro-inflammatory phase (2-8h post inoculation; mainly tumor-necrosis factor α (TNFα) and interleukin-1α driven) and a late anti-inflammatory resolution phase (starting at 24h post inoculation; mainly interleukin-10 and interleukin-4 driven). Interleukin-6 levels correlated with the deterioration of lung function. Lung histology showed maximal changes in terms of inflammation and edema between 24 and 48 h post inoculation. CONCLUSIONS In summary, elevated interleukin-6, high local neutrophil counts and lung edema were found to be the most characteristic signs of the transient period of deterioration of lung function.

Basophils from humans with systemic lupus erythematosus do not express MHC-II.

To the Editor: Recently, Charles et al.1 demonstrated that basophils are able to promote antibody production and aggravate lupus nephritis. Furthermore, they demonstrate that human basophils express major histocompatibility complex class II (MHC-II). The identification of basophils is still problematic owing to the lack of specific basophil markers. Charles et al.1 identified basophils by the expression of the Fce receptor 1a (FceRIa), CD203c, CD123 and CD11b. However, plasmacytoid dendritic cells (pDCs) also express FceRIa (ref. 2) and CD123 (ref. 3) and share the same forward and sideward scatter properties in flow cytometry, rendering their differentiation problematic. Basophils occur together with pDCs in lesional tissues of subjects with human lupus erythematosus4, and pulmonary allergic responses can alter both basophil and pDC responses5. However, because of their similarities, it is difficult to analyze either one of these cell types and to clearly differentiate between the two. To directly compare the phenotype of basophils and pDCs, we characterized basophils and pDCs from eight subjects with systemic lupus erythematosus (SLE, see Supplementary Table 1) by chipcytometry, a technique that allows single-cell analysis of a virtually unlimited number of markers. The specificity is comparable to flow cytometry with a tenfold higher sensitivity6. The methods used in this work are described in the Supplementary Methods. We isolated peripheral blood mononuclear cells (PBMCs) by Ficoll density centrifugation. We depleted T cells (CD3+), monocytes (CD14+), neutrophils (CD16+), B cells (CD20+) and natural killer cells (CD56+) by using a cocktail of antibodies to these proteins and analyzed the remaining cells by chipcytometry6. We identified basophils as CD123+CD304– cells, with variable expression of CD203c and surface-bound IgE (sIgE). We identified pDCs as CD123+CD304+ cells that were negative for CD203c and had low positivity for sIgE (Fig. 1a). In our samples, in contrast to Charles et al.1, we detected expression of MHC-II only on pDCs and not on basophils. We stimulated PBMCs from three subjects with SLE overnight with interleukin-3 (10 ng ml–1) or CpG (0.5 mM) to investigate whether or not MHC-II would be upregulated on the basophils or pDCs. Basophils were still MHC-II–, whereas pDCs upregulated MHC-II expression (Fig. 1b). This upregulation was mediated only by the culture time and not by IL-3 or CpG. In conclusion, we propose that human basophils and pDCs are both CD3–CD14–CD16–CD20–CD56–CD123+, but basophils are MHC-II– CD203cvarCD304–sIgEvar, whereas pDCs are MHC-II+CD203c– CD304+sIgEvar.

Identification and quantification of basophils in the airways of asthmatics following segmental allergen challenge

During asthma attacks, allergens activate sensitized basophils in the lung, thereby aggravating symptoms. Due to the paucity of basophils in bronchial lavage fluid and the lack of specific basophil detection and quantification methods, basophil‐directed research in these samples was hampered in the past. This study aimed to establish and validate a flow cytometry‐based basophil detection and quantification method for human basophils from bronchoalveolar lavage (BAL) and blood as a prerequisite for a better understanding of their pathogenic contribution and subtyping of asthma phenotypes. BAL and blood leukocytes from seasonal asthmatics were analyzed by flow cytometry. Chipcytometry, a highly sensitive single‐cell analysis method, was used to validate the staining panel for basophils. Cell differentials of May–Grünwald–Giemsa‐stained cytospins were used to compare basophil percentages. BAL basophils are identifiable as CD123+HLA‐DR−CD3−CD14−CD19−CD20−CD56− cells in flow cytometrical analysis. Their identity was validated by Chipcytometry. CD203c was highly expressed by BAL basophils, whereas it was expressed at variable levels on blood basophils. The two quantification methods correlated, although more basophils were detected by flow cytometry. Furthermore, the increase in basophil percentages in the lung correlated with the decrease in the basophil percentages in the blood after allergen challenge. We here validated a reliable basophil quantification method, which is independent of the cells activation and degranulation state. The results obtained with this method indicate that basophils are directly recruited from the blood circulation to the airway lumen.

α-NAC–Specific Autoreactive CD8+ T Cells in Atopic Dermatitis Are of an Effector Memory Type and Secrete IL-4 and IFN-γ

Autoreactivity may play a critical role in the chronification of atopic dermatitis (AD). Several studies showed that AD patients produce IgE Abs specific for autoantigens, and we described Th as well as CD8+ T cells specific for the autoallergen Hom s 2, the α-chain of the nascent polypeptide-associated complex (α-NAC). This study aimed to investigate the frequency and inflammatory phenotype of autoallergen-specific CD8+ T cells. CD8+ T cell immunodominant epitopes of α-NAC were mapped by applying prediction softwares, and binding affinity was confirmed by stabilization of empty MHC complexes. MHC class I tetramers were assembled and binding cells were analyzed directly ex vivo by flow cytometry and in terms of single-cell assessment by ChipCytometry. We report significantly elevated numbers of α-NAC–specific peripheral T cells in sensitized patients compared with nonatopic controls. These cells secrete IL-4 and IFN-γ, and surface markers revealed significantly elevated frequencies of circulating terminally differentiated α-NAC–specific CD8+ T cells in patients with AD compared with nonatopic donors. The observed phenotype of α-NAC–specific CD8+ T cells indicates a role in the pathogenesis of AD.

GATA1s induces hyperproliferation of eosinophil precursors in Down syndrome transient leukemia

Transient leukemia (TL) is evident in 5–10% of all neonates with Down syndrome (DS) and associated with N-terminal truncating GATA1 mutations (GATA1s). Here we report that TL-cell clones generate abundant eosinophils in a substantial fraction of patients. Sorted eosinophils from patients with TL and eosinophilia carried the same GATA1s mutations as sorted TL blasts, consistent with their clonal origin. TL blasts exhibited a genetic program characteristic of eosinophils and differentiated along the eosinophil lineage in vitro. Similarly, ectopic expression of Gata1s, but not Gata1, in wild-type CD34+-hematopoietic stem and progenitor cells induced hyperproliferation of eosinophil promyelocytes in vitro. Although GATA1s retained the function of GATA1 to induce eosinophil genes by occupying their promoter regions, GATA1s was impaired in its ability to repress oncogenic MYC and the pro-proliferative E2F transcription network. Chromatin Immunoprecipitation Sequencing (ChIP-seq) indicated reduced GATA1s occupancy at the MYC promoter. Knockdown of MYC, or the obligate E2F-cooperation partner DP1, rescued the GATA1s-induced hyperproliferative phenotype. In agreement, terminal eosinophil maturation was blocked in Gata1Δe2 knockin mice, exclusively expressing Gata1s, leading to accumulation of eosinophil precursors in blood and bone marrow. These data suggest a direct relationship between the N-terminal truncating mutations of GATA1 and clonal eosinophilia in DS patients.

Distinct phenotypic features of neonatal murine macrophages

Neonates rely on their innate immune system. Resident tissue macrophages are considered to be initiators and regulators of the innate immune response and thus, appear to be especially important to neonates. We hypothesized that the phenotype and function of neonatal tissue macrophages differ from their adult counterparts. Peritoneal macrophages from neonatal (<24 h) and adult (6 weeks old) C57BL/6J mice were isolated and analyzed by high‐content chipcytometry. After stimulation for 6 h with LPS (0, 1, 10, 100 ng/mL), macrophage transcriptome was analyzed by microarray and cytokine release was measured using multiparametric bead assays. Antigen presenting capacity was compared by T‐cell stimulation assays. We observed that neonatal murine peritoneal macrophages are characterized by selective lack of expression of F4/80, MHC class II, and costimulatory molecules (CD80, CD86). Furthermore, we found differences in the transcriptome between neonatal and adult macrophages, unstimulated and after LPS stimulation. Although neonatal macrophages showed a significantly increased secretion of proinflammatory cytokines upon LPS stimulation, their potential to induce T‐cell proliferation was significantly reduced. In conclusion, we observed a distinct phenotype of the neonatal macrophage population. The specific functions of this macrophage population could help to understand the excessive inflammatory reactions observed in the very young.