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Abstract Background Forkhead-Box-Protein P3 (FoxP3) is a transcription factor and marker of regulatory T cells, converting naive T cells into Tregs that can downregulate the effector function of other T cells. We previously detected the expression of FoxP3 in retinal pigment epithelial (RPE) cells, forming the outer blood–retina barrier of the immune privileged eye. Methods We investigated the expression, subcellular localization, and phosphorylation of FoxP3 in RPE cells in vivo and in vitro after treatment with various stressors including age, retinal laser burn, autoimmune inflammation, exposure to cigarette smoke, in addition of IL-1β and mechanical cell monolayer destruction. Eye tissue from humans, mouse models of retinal degeneration and rats, and ARPE-19, a human RPE cell line for in vitro experiments, underwent immunohistochemical, immunofluorescence staining, and PCR or immunoblot analysis to determine the intracellular localization and phosphorylation of FoxP3. Cytokine expression of stressed cultured RPE cells was investigated by multiplex bead analysis. Depletion of the FoxP3 gene was performed with CRISPR/Cas9 editing. Results RPE in vivo displayed increased nuclear FoxP3-expression with increases in age and inflammation, long-term exposure of mice to cigarette smoke, or after laser burn injury. The human RPE cell line ARPE-19 constitutively expressed nuclear FoxP3 under non-confluent culture conditions, representing a regulatory phenotype under chronic stress. Confluently grown cells expressed cytosolic FoxP3 that was translocated to the nucleus after treatment with IL-1β to imitate activated macrophages or after mechanical destruction of the monolayer. Moreover, with depletion of FoxP3, but not of a control gene, by CRISPR/Cas9 gene editing decreased stress resistance of RPE cells. Conclusion Our data suggest that FoxP3 is upregulated by age and under cellular stress and might be important for RPE function.

Reconstitution of the T cell repertoire after allogeneic stem cell transplantation is a long and often incomplete process. As a result, reactivation of Epstein-Barr virus (EBV) is a frequent complication that may be treated by adoptive transfer of donor-derived EBV-specific T cells. We generated donor-derived EBV-specific T cells by stimulation with peptides representing defined epitopes covering multiple HLA restrictions. T cells were adoptively transferred to a patient who had developed persisting high titers of EBV after allogeneic stem cell transplantation for angioimmunoblastic T-cell lymphoma (AITL). T cell receptor beta (TCRβ) deep sequencing showed that the T cell repertoire of the patient early after transplantation (day 60) was strongly reduced and only very low numbers of EBV-specific T cells were detectable. Manufacturing and in vitro expansion of donor-derived EBV-specific T cells resulted in enrichment of EBV epitope-specific, HLA-restricted T cells. Monitoring of T cell clonotypes at a molecular level after adoptive transfer revealed that the dominant TCR sequences from peptide-stimulated T cells persisted long-term and established an EBV-specific TCR clonotype repertoire in the host, with many of the EBV-specific TCRs present in the donor. This reconstituted repertoire was associated with immunological control of EBV and with lack of further AITL relapse.

The degree and type of T cell infiltration influence rectal cancer prognosis regardless of classical tumor staging. We asked whether clonal expansion and tumor infiltration are restricted to selected-phenotype T cells; which clones are accessible in peripheral blood; and what the spatial distribution of their target antigens is. From five rectal cancer patients, we isolated paired tumor-infiltrating T cells (TILs) and T cells from unaffected rectum mucosa (TUM) using 13-parameter FACS single cell index sorting. TCRαβ sequences, cytokine, and transcription factor expression were determined with single cell sequencing. TILs and TUM occupied distinct phenotype compartments and clonal expansion predominantly occurred within CD8+ T cells. Expanded TIL clones identified by paired TCRαβ sequencing and exclusively detectable in the tumor showed characteristic PD-1 and TIM-3 expression. TCRβ repertoire sequencing identified 49 out of 149 expanded TIL clones circulating in peripheral blood and 41 (84%) of these were PD-1− TIM-3−. To determine whether clonal expansion of predominantly tumor-infiltrating T cell clones was driven by antigens uniquely presented in tumor tissue, selected TCRs were reconstructed and incubated with cells isolated from corresponding tumor or unaffected mucosa. The majority of clones exclusively detected in the tumor recognized antigen at both sites. In summary, rectal cancer is infiltrated with expanded distinct-phenotype T cell clones that either i) predominantly infiltrate the tumor, ii) predominantly infiltrate the unaffected mucosa, or iii) overlap between tumor, unaffected mucosa, and peripheral blood. However, the target antigens of predominantly tumor-infiltrating TIL clones do not appear to be restricted to tumor tissue.

Multinucleated giant cells (MGCs) form by fusion of macrophages and are presumed to contribute to the removal of debris from tissues. In a systematic in vitro analysis, we show that IL-4-induced MGCs phagocytosed large and complement-opsonized materials more effectively than their unfused M2 macrophage precursors. MGC expression of complement receptor 4 (CR4) was increased, but it functioned primarily as an adhesion integrin. In contrast, although expression of CR3 was not increased, it became functionally activated during fusion and was located on the extensive membrane ruffles created by excess plasma membrane arising from macrophage fusion. The combination of increased membrane area and activated CR3 specifically equips MGCs to engulf large complement-coated targets. Moreover, we demonstrate these features in vivo in the recently described complement-dependent therapeutic elimination of systemic amyloid deposits by MGCs. MGCs are evidently more than the sum of their macrophage parts.

Summary: Branched-chain amino acid (BCAA) metabolism is linked to glucose homeostasis, but the underlying signaling mechanisms are unclear. We find that gluconeogenesis is reduced in mice deficient of Ppm1k, a positive regulator of BCAA catabolism, which protects against obesity-induced glucose intolerance. Accumulation of branched-chain keto acids (BCKAs) inhibits glucose production in hepatocytes. BCKAs suppress liver mitochondrial pyruvate carrier (MPC) activity and pyruvate-supported respiration. Pyruvate-supported gluconeogenesis is selectively suppressed in Ppm1k-deficient mice and can be restored with pharmacological activation of BCKA catabolism by BT2. Finally, hepatocytes lack branched-chain aminotransferase that alleviates BCKA accumulation via reversible conversion between BCAAs and BCKAs. This renders liver MPC most susceptible to circulating BCKA levels hence a sensor of BCAA catabolism.

In hypertrophied and failing hearts, fuel metabolism is reprogrammed to increase glucose metabolism, especially glycolysis. This metabolic shift favors biosynthetic function at the expense of ATP production. Mechanisms responsible for the switch are poorly understood. We found that inhibitory factor 1 of the mitochondrial FoF1-ATP synthase (ATPIF1), a protein known to inhibit ATP hydrolysis by the reverse function of ATP synthase during ischemia, was significantly upregulated in pathological cardiac hypertrophy induced by pressure overload, myocardial infarction, or α-adrenergic stimulation. Chemical cross-linking mass spectrometry analysis of hearts hypertrophied by pressure overload suggested that increased expression of ATPIF1 promoted the formation of FoF1-ATP synthase nonproductive tetramer. Using ATPIF1 gain- and loss-of-function cell models, we demonstrated that stalled electron flow due to impaired ATP synthase activity triggered mitochondrial ROS generation, which stabilized HIF1α, leading to transcriptional activation of glycolysis. Cardiac-specific deletion of ATPIF1 in mice prevented the metabolic switch and protected against the pathological remodeling during chronic stress. These results uncover a function of ATPIF1 in nonischemic hearts, which gives FoF1-ATP synthase a critical role in metabolic rewiring during the pathological remodeling of the heart.

Hypertrophic cardiomyocytes switch their metabolism from fatty acid oxidation to glucose use, but the functional role of this change is unclear. Here the authors show that high intracellular glucose inhibits the degradation of branched-chain amino acids, which is required for the activation of pro-growth mTOR signaling.

Abstract Members of the S100 protein family have been reported to function as endogenous danger signals (alarmins) playing an active role in tissue inflammation and repair when released from necrotic cells. Here, we investigated the role of S100A1, the S100 isoform with highest abundance in cardiomyocytes, when released from damaged cardiomyocytes during myocardial infarction (MI). Patients with acute MI showed significantly increased S100A1 serum levels. Experimental MI in mice induced comparable S100A1 release. S100A1 internalization was observed in cardiac fibroblasts (CFs) adjacent to damaged cardiomyocytes. In vitro analyses revealed exclusive S100A1 endocytosis by CFs, followed by Toll‐like receptor 4 (TLR4)‐dependent activation of MAP kinases and NF‐κB. CFs exposed to S100A1 assumed an immunomodulatory and anti‐fibrotic phenotype characterized i.e. by enhanced intercellular adhesion molecule‐1 (ICAM1) and decreased collagen levels. In mice, intracardiac S100A1 injection recapitulated these transcriptional changes. Moreover, antibody‐mediated neutralization of S100A1 enlarged infarct size and worsened left ventricular functional performance post‐MI. Our study demonstrates alarmin properties for S100A1 from necrotic cardiomyocytes. However, the potentially beneficial role of extracellular S100A1 in MI‐related inflammation and repair warrants further investigation.