Marcin Łyszkiewicz

Novel reporter mouse reveals constitutive and inflammatory expression of IFN-beta in vivo.

Type I IFN is a major player in innate and adaptive immune responses. Besides, it is involved in organogenesis and tumor development. Generally, IFN responses are amplified by an autocrine loop with IFN-β as the priming cytokine. However, due to the lack of sensitive detection systems, where and how type I IFN is produced in vivo is still poorly understood. In this study, we describe a luciferase reporter mouse, which allows tracking of IFN-β gene induction in vivo. Using this reporter mouse, we reveal strong tissue-specific induction of IFN-β following infection with influenza or La Crosse virus. Importantly, this reporter mouse also allowed us to visualize that IFN-β is expressed constitutively in several tissues. As suggested before, low amounts of constitutively produced IFN might maintain immune cells in an activated state ready for a timely response to pathogens. Interestingly, thymic epithelial cells were the major source of IFN-β under noninflammatory conditions. This relatively high constitutive expression was controlled by the NF Aire and might influence induction of tolerance or T cell development.

Critical role for miR-181a/b-1 in agonist selection of invariant natural killer T cells

T-cell receptor (TCR) signal strength determines selection and lineage fate at the CD4+CD8+ double-positive stage of intrathymic T-cell development. Members of the miR-181 family constitute the most abundantly expressed microRNA at this stage of T-cell development. Here we show that deletion of miR-181a/b-1 reduced the responsiveness of double-positive thymocytes to TCR signals and virtually abrogated early invariant natural killer T (iNKT) cell development, resulting in a dramatic reduction in iNKT cell numbers in thymus as well as in the periphery. Increased concentrations of agonist ligand rescued iNKT cell development in miR-181a/b-1−/− mice. Our results define a critical role of miR-181a/b-1 in early iNKT cell development and show that miR-181a/b-1 sets a TCR signaling threshold for agonist selection.

Multiple extrathymic precursors contribute to T-cell development with different kinetics.

T-cell development in the thymus depends on continuous supply of T-cell progenitors from bone marrow (BM). Several extrathymic candidate progenitors have been described that range from multipotent cells to lymphoid cell committed progenitors and even largely T-lineage committed precursors. However, the nature of precursors seeding the thymus under physiologic conditions has remained largely elusive and it is not known whether there is only one physiologic T-cell precursor population or many. Here, we used a competitive in vivo assay based on depletion rather than enrichment of classes of BM-derived precursor populations, thereby only minimally altering physiologic precursor ratios to assess the contribution of various extrathymic precursors to T-lineage differentiation. We found that under these conditions multiple precursors, belonging to both multipotent progenitor (MPP) and common lymphoid progenitor (CLP) subsets have robust T-lineage potential. However, differentiation kinetics of different precursors varied considerably, which might ensure continuous thymic output despite gated importation of extrathymic precursors. In conclusion, our data suggest that the thymus functions to impose T-cell fate on any precursor capable of filling the limited number of progenitor niches.

Somatic hypermutation in peritoneal B1b cells.

Murine B1 cells have been shown to be able to switch to IgA in vitro. In agreement, we could demonstrate in the peritoneum of mice the presence of IgA producing B1 cells. Interestingly, enzyme-linked immunospot assays of lipopolysaccharide stimulated cultures revealed that only the B1b cell subpopulation contained high numbers of such cells while IgA producing B cells were rare amongst the B2 and B1a cell populations. This was confirmed by RT-PCR on sorted peritoneal B cell subpopulations. In addition, the variable regions associated with IgA of peritoneal B1b cells displayed extensive variation due to somatic hypermutation. In contrast, mutations were found only at low frequencies in VH regions associated with IgM of both B1 cell populations. Thus, peritoneal B1b cells display many similarities to B2 cells. This finding is consistent with the idea of a layered immune system in which peritoneal B1a and splenic follicular B2 cells appear at the two extremes and peritoneal B1b and B2 cells represent intermediates.

Absence of IFN-β Impairs Antigen Presentation Capacity of Splenic Dendritic Cells via Down-Regulation of Heat Shock Protein 70

Type I IFNs play a key role in linking the innate and adaptive arms of the immune system. Although produced rapidly in response to pathogens, IFNs are also produced at low levels in the absence of infection. In the present study, we demonstrate that constitutively produced IFNs are necessary in vivo to maintain dendritic cells in an “Ag presentation-competent” state. Conventional dendritic cells (cDCs) isolated from spleens of IFN-β or IFNAR-deficient mice exhibit a highly impaired ability to present Ag and activate naive T cells. Microarray analysis of mRNA isolated from IFN-β−/− and IFNAR−/− cDCs revealed diminished expression of two genes that encoded members of the heat shock protein 70 (Hsp70) family. Consistent with this observation, pharmacological inhibition of Hsp70 in cDCs from wild-type mice impaired their T cell stimulatory capacity. Similarly, the Ag presentation ability of splenic cDCs isolated from Hsp70.1/3−/− mice was also severely impaired in comparison to wild-type cDCs. Thus, constitutive IFN-β expression regulates Hsp70 levels to help maintain dendritic cells in a competent state for efficient priming of effector T cells in vivo.

Multicongenic fate mapping quantification of dynamics of thymus colonization

Ziętara et al demonstrate with multicongenic fate mapping that thymus seeding is directly restricted to the duration of niche occupancy rather than long-range effects.

CCR7-mediated migration in the thymus controls γδ T-cell development

αβ T‐cell development and selection proceed while thymocytes successively migrate through distinct regions of the thymus. For γδ T cells, the interplay of intrathymic migration and cell differentiation is less well understood. Here, we crossed C‐C chemokine receptor (CCR)7‐deficient (Ccr7−/−) and CCR9‐deficient mice (Ccr9−/−) to mice with a TcrdH2BeGFP reporter background to investigate the impact of thymic localization on γδ T‐cell development. γδ T‐cell frequencies and numbers were decreased in CCR7‐deficient and increased in CCR9‐deficient mice. Transfer of CCR7‐ or CCR9‐deficient BM into irradiated C57BL/6 WT recipients reproduced these phenotypes, pointing toward cell‐intrinsic migration defects. Monitoring recent thymic emigrants by intrathymic labeling allowed us to identify decreased thymic γδ T‐cell output in CCR7‐deficient mice. In vitro, CCR7‐deficient precursors showed normal γδ T‐cell development. Immunohistology revealed that CCR7 and CCR9 expression was important for γδ T‐cell localization within thymic medulla or cortex, respectively. However, γδ T‐cell motility was unaltered in CCR7‐ or CCR9‐deficient thymi. Together, our results suggest that proper intrathymic localization is important for normal γδ T‐cell development.

ICOS‐dependent stimulation of NKT cells by marginal zone B cells

Marginal zone (MZ) B cells express high levels of CD1d molecules. In accordance, MZ B cells, like splenic conventional DCs (cDCs), efficiently trigger NKT‐cell proliferation. Importantly, MZ B cells exclusively induced production of IL‐4 and IL‐13 by such cells whereas cDCs induced robust production of mainly IFN‐γ. NKT‐cell proliferation, IL‐4 and IL‐13 production induced by MZ B cells were dependent on ICOS/ICOS ligand interaction while IFN‐γ and IL‐17 induction by cDCs required glucocorticoid‐induced TNF receptor/glucocorticoid‐induced TNF receptor ligand interplay. Our data illustrate that both MZ B cells and cDCs act as efficient APCs for NKT cells and might differentially influence the quality of the subsequent immune response.

T Cell Development by the Numbers

T cells are continually generated in the thymus in a highly dynamic process comprising discrete steps of lineage commitment, T cell receptor (TCR) gene rearrangement, and selection. These steps are linked to distinct rates of proliferation, survival, and cell death, but a quantitative picture of T cell development is only beginning to emerge. Here we summarize recent technical advances, including genetic fate mapping, barcoding, and molecular timers, that have allowed the implementation of computational models to quantify developmental dynamics in the thymus. Coupling new techniques with mathematical models has recently resulted in the emergence of new paradigms in early hematopoiesis and might similarly open new perspectives on T cell development.

Immunoglobulins drive terminal maturation of splenic dendritic cells

Nature and physiological status of antigen-presenting cells, such as dendritic cells DCs, are decisive for the immune reactions elicited. Multiple factors and cell interactions have been described that affect maturation of DCs. Here, we show that DCs arising in the absence of immunoglobulins (Ig) in vivo are impaired in cross-presentation of soluble antigen. This deficiency was due to aberrant cellular targeting of antigen to lysosomes and its rapid degradation. Function of DCs could be restored by transfer of Ig irrespective of antigen specificity and isotype. Modulation of cross-presentation by Ig was inhibited by coapplication of mannan and, thus, likely to be mediated by C-type lectin receptors. This unexpected dependency of splenic DCs on Ig to cross-present antigen provides insights into the interplay between cellular and humoral immunity and the immunomodulatory capacity of Ig.