Correction to: High-dimensional single cell mass cytometry analysis of the murine hematopoietic system reveals signatures induced by ageing and physiological pathogen challenges.

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High-dimensional single cell mass cytometry analysis of the murine hematopoietic system reveals signatures induced by ageing and physiological pathogen challenges.

BACKGROUND: Immune ageing is a result of repetitive microbial challenges along with cell intrinsic or systemic changes occurring during ageing. Mice under 'specific-pathogen-free' (SPF) conditions are frequently used to assess immune ageing in long-term experiments. However, physiological pathogenic challenges are reduced in SPF mice. The question arises to what extent murine experiments performed under SPF conditions are suited to analyze immune ageing in mice and serve as models for human immune ageing. Our previous comparisons of same aged mice with different microbial exposures, unambiguously identified distinct clusters of immune cells characteristic for numerous previous pathogen encounters in particular in pet shop mice. RESULTS: We here performed single cell mass cytometry assessing splenic as secondary and bone marrow as primary lymphoid organ-derived leukocytes isolated from young versus aged SPF mice in order to delineate alterations of the murine hematopoietic system induced during ageing. We then compared immune clusters from young and aged SPF mice to pet shop mice in order to delineate alterations of the murine hematopoietic system induced by physiological pathogenic challenges and those caused by cell intrinsic or systemic changes during ageing. Notably, distinct immune signatures were similarly altered in both pet shop and aged SPF mice in comparison to young SPF mice, including increased frequencies of memory T lymphocytes, effector-cytokine producing T cells, plasma cells and mature NK cells. However, elevated frequencies of CD4+ T cells, total NK cells, granulocytes, pDCs, cDCs and decreased frequencies of naïve B cells were specifically identified only in pet shop mice. In aged SPF mice specifically the frequencies of splenic IgM+ plasma cells, CD8+ T cells and CD4+ CD25+ Treg were increased as compared to pet shop mice and young mice. CONCLUSIONS: Our study dissects firstly how ageing impacts both innate and adaptive immune cells in primary and secondary lymphoid organs. Secondly, it partly distinguishes murine intrinsic immune ageing alterations from those induced by physiological pathogen challenges highlighting the importance of designing mouse models for their use in preclinical research including vaccines and immunotherapies.

Blockade of the costimulatory CD28-B7 family signal axis enables repeated application of AAV8 gene vectors.

Adeno-associated virus serotype 8 (AAV8) gene therapy has shown efficacy in several clinical trials and is considered a highly promising technology to treat monogenic diseases such as hemophilia A and B. However, a major drawback of AAV8 gene therapy is that it can be applied only once because anti-AAV8 immunity develops after the first treatment. Readministration may be required in patients who are expected to need redosing, eg, due to organ growth, or to boost suboptimal expression levels, but no redosing protocol has been established. We have developed a preventive immune-suppressive protocol for a human factor IX (FIX) vector with an intended dose of ~5 × 1011  vg/kg that inhibits the development of anti-AAV8 neutralizing-antibody (NAb) responses and anti-AAV8 T-cell responses using CTLA4-IgG (abatacept). In a preclinical model, transient treatment with abatacept during initial human FIX gene therapy efficiently inhibited the generation of AAV8-specific cellular and humoral responses, and thus permitted redosing of FIX. Furthermore, our data suggest that by suppression of anti-AAV8 NAb responses after the second higher dose (4 × 1012  vg/kg) this protocol can be used to enable redosing up to such high doses. An additional advantage of CTLA4-IgG blocking CD28-mediated signals is its potential suppression of AAV8-specific cytotoxic CD8 T-cell responses, which are believed to kill transduced hepatocytes and might interfere with a successful readministration. Redosing protocols using approved drugs would be beneficial for patients because they could effortlessly be applied in clinical trials and enable safe and efficient treatment options for patients undergoing AAV8 gene therapy.

Wild immunology assessed by multidimensional mass cytometry.

A great part of our knowledge on mammalian immunology has been established in laboratory settings. The use of inbred mouse strains enabled controlled studies of immune cell and molecule functions in defined settings. These studies were usually performed in specific-pathogen free (SPF) environments providing standardized conditions. In contrast, mammalians including humans living in their natural habitat are continuously facing pathogen encounters throughout their life. The influences of environmental conditions on the signatures of the immune system and on experimental outcomes are yet not well defined. Thus, the transferability of results obtained in current experimental systems to the physiological human situation has always been a matter of debate. Studies elucidating the diversity of "wild immunology" imprintings in detail and comparing it with those of "clean" lab mice are sparse. Here, we applied multidimensional mass cytometry to dissect phenotypic and functional differences between distinct groups of laboratory and pet shop mice as a source for "wild mice". For this purpose, we developed a 31-antibody panel for murine leukocyte subsets identification and a 35-antibody panel assessing various cytokines. Established murine leukocyte populations were easily identified and diverse immune signatures indicative of numerous pathogen encounters were classified particularly in pet shop mice and to a lesser extent in quarantine and non-SPF mice as compared to SPF mice. In addition, unsupervised analysis identified distinct clusters that associated strongly with the degree of pathogenic priming, including increased frequencies of activated NK cells and antigen-experienced B- and T-cell subsets. Our study unravels the complexity of immune signatures altered under physiological pathogen challenges and highlights the importance of carefully adapting laboratory settings for immunological studies in mice, including drug and therapy testing. © 2016 International Society for Advancement of Cytometry.

CD40L expression by CD4+ but not CD8+ T cells regulates antiviral immune responses in acute LCMV infection in mice.

CD40-CD40 ligand (CD40L) signaling plays multiple indispensable roles in cellular and humoral immunity. Impaired memory T-cell responses in the absence of CD40L have been well documented, but the requirement of this interaction for efficient priming of CD8+ T cells especially under inflammatory conditions has been under debate. In contrast to previous publications, we report here that virus-specific CD8+ T-cell responses as well as viral clearance are affected not only in the memory but also in the effector phase in CD40L-/- mice infected with lymphocytic choriomeningitis virus (LCMV) Armstrong strain. Interestingly, a considerable part of the LCMV-specific effector and memory T cells consists of CD40L+ CD8+ T cells. However, deficiency of CD40L in CD8+ T cells did influence neither the quantity nor the quality of primary T-cell responses in LCMV infection. Virus-specific CD8+ T cells in conditional knockout mice, with a selective deletion of the CD40L in CD8+ T cells, were fully functional regarding cytokine production and efficient pathogen clearance. Thus, our results unambiguously demonstrate that while CD40L is critical to generate effective primary CD8+ T-cell responses also under inflammatory conditions, CD40L expression by CD8+ T cells themselves is dispensable in acute LCMV infection.

Human RORγt(+)CD34(+) cells are lineage-specified progenitors of group 3 RORγt(+) innate lymphoid cells.

Group 3 innate lymphoid cells (ILC3s) are defined by the expression of the transcription factor RORγt, which is selectively required for their development. The lineage-specified progenitors of ILC3s and their site of development after birth remain undefined. Here we identified a population of human CD34(+) hematopoietic progenitor cells (HPCs) that express RORγt and share a distinct transcriptional signature with ILC3s. RORγt(+)CD34(+) HPCs were located in tonsils and intestinal lamina propria (LP) and selectively differentiated toward ILC3s. In contrast, RORγt(-)CD34(+) HPCs could differentiate to become either ILC3s or natural killer (NK) cells, with differentiation toward ILC3 lineage determined by stem cell factor (SCF) and aryl hydrocarbon receptor (AhR) signaling. Thus, we demonstrate that in humans RORγt(+)CD34(+) cells are lineage-specified progenitors of IL-22(+) ILC3s and propose that tonsils and intestinal LP, which are enriched both in committed precursors and mature ILC3s, might represent preferential sites of ILC3 lineage differentiation.

CD40L expression permits CD8+ T cells to execute immunologic helper functions.

CD8(+) T cells play an essential role in immunity against intracellular pathogens, with cytotoxicity being considered their major effector mechanism. However, we here demonstrate that a major part of central and effector memory CD8(+) T cells expresses CD40L, one key molecule for CD4(+) T-cell-mediated help. CD40L(+) CD8(+) T cells are detectable among human antigen-specific immune responses, including pathogens such as influenza and yellow fever virus. CD40L(+) CD8(+) T cells display potent helper functions in vitro and in vivo, such as activation of antigen-presenting cells, and exhibit a cytokine expression signature similar to CD4(+) T cells and unrelated to cytotoxic CD8(+) T cells. The broad occurrence of CD40L(+) CD8(+) T cells in cellular immunity implicates that helper functions are not only executed by major histocompatibility complex (MHC) class II-restricted CD4(+) helper T cells but are also a common feature of MHC class I-restricted CD8(+) T cell responses. Due to their versatile functional capacities, human CD40L(+) CD8(+) T cells are promising candidate cells for immune therapies, particularly when CD4(+) T-cell help or pathogen-associated molecular pattern signals are limited.

A converse 4-1BB and CD40 ligand expression pattern delineates activated regulatory T cells (Treg) and conventional T cells enabling direct isolation of alloantigen-reactive natural Foxp3+ Treg.

Natural regulatory T cells (nTreg) play a central role in the induction and maintenance of immunological tolerance. Experimental transplant models and recent clinical trials demonstrate that nTreg can control alloreactivity. To upgrade Treg-based cell therapies to a selective suppression of undesired immune reactions, only the transfer of Ag-specific nTreg represents the appropriate therapeutic option. However, Ag-specific nTreg are present at extremely low frequencies in the periphery, and so far appropriate surface markers for their precise identification are missing. In this study, we demonstrate that activated nTreg and activated conventional T cells differ in their 4-1BB and CD40 ligand (CD40L) expression signatures, allowing a clear dissection from each other. Based on the expression of 4-1BB and absence of CD40L expression, human alloantigen-reactive Foxp3(+) nTreg can be directly isolated from MLR cultures with high purity. Alloantigen-reactive 4-1BB(+)CD40L(-) nTreg were characterized by a completely demethylated Treg-specific demethylated region and showed alloantigen-specific suppressive properties superior to polyclonal Treg. Importantly, isolated 4-1BB(+)CD40L(-) nTreg maintain the nTreg phenotype and alloantigen-reactivity after in vitro expansion. Our results offer the possibility to simultaneously analyze Ag-specific nTreg and conventional T cells, and to establish cellular therapies with Ag-specific nTreg aiming at a specific inhibition of unwanted immunity.