Regulatory T (Treg) cells maintain immune homeostasis and prevent inflammatory and autoimmune responses. During development, thymocytes bearing a moderately self-reactive T cell receptor (TCR) can be selected to become Treg cells. Several observations suggest that also in the periphery mature Treg cells continuously receive self-reactive TCR signals. However, the importance of this inherent autoreactivity for Treg cell biology remains poorly defined. To address this open question, we genetically ablated the TCR of mature Treg cells in vivo. These experiments revealed that TCR-induced Treg lineage-defining Foxp3 expression and gene hypomethylation were uncoupled from TCR input in mature Treg cells. However, Treg cell homeostasis, cell-type-specific gene expression and suppressive function critically depend on continuous triggering of their TCR.

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Upconversion nanoparticles (UCNPs) convert near-infrared into visible light at much lower excitation densities than those used in classic two-photon absorption microscopy. Here, we engineered <50 nm UCNPs for application as efficient lanthanide resonance energy transfer (LRET) donors inside living cells. By optimizing the dopant concentrations and the core-shell structure for higher excitation densities, we observed enhanced UCNP emission as well as strongly increased sensitized acceptor fluorescence. For the application of these UCNPs in complex biological environments, we developed a biocompatible surface coating functionalized with a nanobody recognizing green fluorescent protein (GFP). Thus, rapid and specific targeting to GFP-tagged fusion proteins in the mitochondrial outer membrane and detection of protein interactions by LRET in living cells was achieved.

IgA is prominently secreted at mucosal surfaces and coats a fraction of the commensal microbiota, a process that is critical for intestinal homeostasis. However, the mechanisms of IgA induction and the molecular targets of these antibodies remain poorly understood, particularly in humans. Here, we demonstrate that microbiota from a subset of human individuals encode two protein “superantigens” expressed on the surface of commensal bacteria of the family Lachnospiraceae such as Ruminococcus gnavus that bind IgA variable regions and stimulate potent IgA responses in mice. These superantigens stimulate B cells expressing human VH3 or murine VH5/6/7 variable regions and subsequently bind their antibodies, allowing these microbial organisms to become highly coated with IgA in vivo. These findings demonstrate a previously unappreciated role for commensal superantigens in host-microbiota interactions. Furthermore, as superantigen-expressing strains show an uneven distribution across human populations, they should be systematically considered in studies evaluating human B cell responses and microbiota during homeostasis and disease.

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Maintaining immune tolerance requires the production of Foxp3-expressing regulatory T (Treg) cells in the thymus. Activation of NF-κB transcription factors is critically required for Treg cell development, partly via initiating Foxp3 expression. NF-κB activation is controlled by a negative feedback regulation through the ubiquitin editing enzyme A20, which reduces proinflammatory signaling in myeloid cells and B cells. In naive CD4+ T cells, A20 prevents kinase RIPK3-dependent necroptosis. Using mice deficient for A20 in T lineage cells, we show that thymic and peripheral Treg cell compartments are quantitatively enlarged because of a cell-intrinsic developmental advantage of A20-deficient thymic Treg differentiation. A20-deficient thymic Treg cells exhibit reduced dependence on IL-2 but unchanged rates of proliferation and apoptosis. Activation of the NF-κB transcription factor RelA was enhanced, whereas nuclear translocation of c-Rel was decreased in A20-deficient thymic Treg cells. Furthermore, we found that the increase in Treg cells in T cell–specific A20-deficient mice was already observed in CD4+ single-positive CD25+ GITR+ Foxp3− thymic Treg cell progenitors. Treg cell precursors expressed high levels of the tumor necrosis factor receptor superfamily molecule GITR, whose stimulation is closely linked to thymic Treg cell development. A20-deficient Treg cells efficiently suppressed effector T cell–mediated graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, suggesting normal suppressive function. Holding thymic production of natural Treg cells in check, A20 thus integrates Treg cell activity and increased effector T cell survival into an efficient CD4+ T cell response.

On the basis of a protein cage scaffold, we have systematically explored intracellular application of nanoparticles for single molecule studies and discovered that recognition by the autophagy machinery plays a key role for rapid metabolism in the cytosol. Intracellular stealth nanoparticles were achieved by heavy surface PEGylation. By combination with a generic approach for nanoparticle monofunctionalization, efficient labeling of intracellular proteins with high fidelity was accomplished, allowing unbiased long-term tracking of proteins in the outer mitochondrial membrane.

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