B cells and macrophages fail to induce transcriptional enhancer activity of the TSDR.

<p>Dual luciferase assays were performed after transfecting reporter plasmids carrying the indicated inserts or an empty pGL3 vector (EV) into RLM-11 cells (T cell line), A20 cells (B cell line) or RAW 264.7 cells (macrophage cell line). Three hrs (RLM-11, A20) or 20 hrs (RAW 264.7) after transfection, cells were stimulated for 16 hrs with PMA/iono (RLM-11, A20) or for 14 hrs with LPS/IFN-γ (RAW 264.7), followed by measurement of luciferase activities (mean±SD, n = 3). Data are representative of two to four independent experiments.</p

Degradation of IκBα is not required for TSDR enhancer activity.

<p>Luciferase plasmids integrating either the NF-κB-RE or TSDR-FoxPro were co-transfected with either an empty vector or with a vector encoding the super-repressor, a non-degradable form of IκBα, into RLM-11 cells. Dual luciferase assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088318#pone-0088318-g001" target="_blank">Figure 1</a> and unstimulated cells served as controls. Luciferase activities are shown as percent of empty vector controls and standard deviations of performed triplicates are shown. One representative experiment out of at least two independent experiments is depicted.</p

The postulated NF-κB binding site of the TSDR is not transcriptionally responsive to activated NF-κB.

<p>(<b>A</b>) RLM-11 cells were stimulated with PMA/iono for indicated time periods and applied to subcellular fractionation. Nuclear and cytoplasmic extracts were analyzed by Western blotting using the indicated antibodies. p44/42 and lamin B served as loading and purity controls for cytoplasmic and nuclear fractions, respectively. (<b>B</b>) A luciferase reporter plasmid integrating the NF-κB-RE was transfected into RLM-11 cells and dual luciferase assays were performed in triplicates as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088318#pone-0088318-g001" target="_blank">Figure 1</a>. Mean luciferase activity is shown as fold increase to unstimulated control. Results are representative of four independent experiments. (<b>C</b>) A schematic view on the first part of the <i>Foxp3</i> gene locus is depicted. White boxes indicate untranslated exons, the first translated exon is indicated in black. Evolutionary conserved non-coding sequences (CNS) are indicated in grey. The distended region of the TSDR includes the previously described NF-κB binding site (black frame), which is flanked by the seventh CpG motif (underlined) of the TSDR. (<b>D</b>) A tandem of five repetitive sequences of the putative NF-κB binding site was inserted into the pCpGL luciferase reporter plasmid upstream of the <i>EF</i> promoter (tandem-EFPro). Dual luciferase assays were performed as described in (B) using pCpGL-TSDR-EFPro as a positive control. Data represent one out of two independent experiments.</p

c-Rel^−/− Tregs show a stable phenotype.

<p>(<b>A</b>) CD4⁺CD25^hi Tregs and CD4⁺CD25⁻ Tconv were isolated from wild-type (WT) or c-Rel^−/− mice. Genomic DNA was isolated and subjected to bisulfite sequencing in order to determine the methylation status of CpG dinucleotides within the TSDR. (<b>B</b>) CD4⁺CD8⁻CD62L^hiCD25^hi Tregs from spleen and lymph nodes of c-Rel^−/− or WT mice were sorted and an aliquot was analyzed for Foxp3 expression by flow cytometry (top panel). Cells were cultured in the presence of IL-2 and stimulated by plate-bound α-CD3/CD28 for six days followed by flow cytometric analysis of Foxp3 expression. Cells depicted were pregated to viable CD4⁺ T cells. Results represent one out of two independent experiments.</p

Kinase activity of IKKα and IKKβ is largely dispensable for TSDR enhancer activity.

<p>(<b>A</b>) Luciferase plasmids encoding NF-κB-RE or TSDR-FoxPro were co-transfected with plasmids encoding kinase dead (KD) or wild-type (WT) forms of IκB kinase α and β (IKKα and IKKβ) into RLM-11 cells. Cells were cultured for one day allowing efficient protein expression before cells were stimulated overnight with PMA/iono and dual luciferase assays were performed. Luciferase activities are given as percent of luciferase activity of WT samples and standard deviations were calculated from three replicates. (<b>B</b>) Dual luciferase assays as described in (A) were performed co-transfecting the indicated luciferase constructs with a plasmid encoding the constitutively active form of IKKβ (IKK-CA) or empty vector as control (mean±SD, n = 3). One representative out of three independent experiments is shown.</p

Intracellular <i>Staphylococcus aureus</i> eludes selective autophagy by activating a host cell kinase

<p>Autophagy, a catabolic pathway of lysosomal degradation, acts not only as an efficient recycle and survival mechanism during cellular stress, but also as an anti-infective machinery. The human pathogen <i>Staphylococcus aureus</i> (<i>S. aureus</i>) was originally considered solely as an extracellular bacterium, but is now recognized additionally to invade host cells, which might be crucial for persistence. However, the intracellular fate of <i>S. aureus</i> is incompletely understood. Here, we show for the first time induction of selective autophagy by <i>S. aureus</i> infection, its escape from autophagosomes and proliferation in the cytoplasm using live cell imaging. After invasion, <i>S. aureus</i> becomes ubiquitinated and recognized by receptor proteins such as SQSTM1/p62 leading to phagophore recruitment. Yet, <i>S. aureus</i> evades phagophores and prevents further degradation by a MAPK14/p38α MAP kinase-mediated blockade of autophagy. Our study demonstrates a novel bacterial strategy to block autophagy and secure survival inside the host cell.</p

sj-docx-1-tan-10.1177_17562864221141505 – Supplemental material for Preserved T-cell response in anti-CD20-treated multiple sclerosis patients following SARS-CoV-2 vaccination

Supplemental material, sj-docx-1-tan-10.1177_17562864221141505 for Preserved T-cell response in anti-CD20-treated multiple sclerosis patients following SARS-CoV-2 vaccination by Simon Faissner, Neele Heitmann, Ricarda Rohling, Ulas Ceylan, Marielena Bongert, Carlos Plaza-Sirvent, Corinna Marheinecke, Xiomara Pedreiturria, Ilya Ayzenberg, Kerstin Hellwig, Ingo Schmitz, Stephanie Pfaender and Ralf Gold in Therapeutic Advances in Neurological Disorders</p

DataSheet_1_Immune response in ofatumumab treated multiple sclerosis patients after SARS-CoV-2 vaccination.pdf

ObjectiveThe pandemic induced by SARS-CoV-2 has huge implications for patients with immunosuppression that is caused by disorders or specific treatments. Especially approaches targeting B cells via anti-CD20 therapy are associated with impaired humoral immune response but sustained cellular immunity. Ofatumumab is a human anti-CD20 directed antibody applied in low dosages subcutaneously, recently licensed for Multiple Sclerosis (MS). Effects of early ofatumumab treatment on alterations of immune cell composition and immune response towards SARS-CoV-2 are incompletely understood.MethodsWe here investigated immune cell alterations in early ofatumumab (Ofa) treated patients and effects on humoral (titer, neutralization capacity against wild type, Delta and Omicron) and cellular immune responses in Ofa treated MS patients following a third vaccination against SARS-CoV-2 compared to healthy controls.ResultsWe show that a mean treatment duration of three months in the Ofa group led to near complete B cell depletion in line with altered composition of certain CD4+ T cell subpopulations such as enhanced frequencies of naive and a decrease of non-suppressive regulatory T cells (Tregs). Titer and neutralization capacity against SARS-CoV-2 variants was impaired while cellular immune response was sustained, characterized by a strong T helper 1 profile (Th1).InterpretationIn summary, low dosage ofatumumab treatment elicits sustained depletion of B cells in line with alterations of immune cells, mainly Tregs. This is associated with impaired humoral immune response towards SARS-CoV-2 vaccination but preserved, Th1 driven cellular immunity adding crucial information regarding early effects of low dosage anti-CD20 therapy on humoral and cellular immunity.</p

DataSheet_1_Impact of SARS-CoV-2 vaccination on systemic immune responses in people living with HIV.pdf

Despite the development of vaccines, which protect healthy people from severe and life-threatening Covid-19, the immunological responses of people with secondary immunodeficiencies to these vaccines remain incompletely understood. Here, we investigated the humoral and cellular immune responses elicited by mRNA-based SARS-CoV-2 vaccines in a cohort of people living with HIV (PLWH) receiving anti-retroviral therapy. While antibody responses in PLWH increased progressively after each vaccination, they were significantly reduced compared to the HIV-negative control group. This was particularly noteworthy for the Delta and Omicron variants. In contrast, CD4+ Th cell responses exhibited a vaccination-dependent increase, which was comparable in both groups. Interestingly, CD4+ T cell activation negatively correlated with the CD4 to CD8 ratio, indicating that low CD4+ T cell numbers do not necessarily interfere with cellular immune responses. Our data demonstrate that despite the lower CD4+ T cell counts SARS-CoV-2 vaccination results in potent cellular immune responses in PLWH. However, the reduced humoral response also provides strong evidence to consider PLWH as vulnerable group and suggests subsequent vaccinations being required to enhance their protection against COVID-19.</p