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Additional file 1: Figure S1. Indirect immune fluorescence microscopy in murine heterozygous Gla+/- and homozygous Gla-/- female DRG, and viability assay in BV-2 cells. (A) Representative MP images of female DRG using anti-Gb3 (CD77), anti-CD68 and anti-CD206. Scale bar 50 µm. (B) Quantification of stained DRG sections, 42 weeks Gla+/- n=3 and Gla-/- n=3 mice. Statistical significance was assessed using Mann–Whitney U test. (C) Viability test of BV-2 cells in Lyso-Gb3 and Gb3 (1% DMSO was used as vehicle). Values are given as mean ± SEM. Figure S2. Gating strategy. Following doublet and dead cell exclusion using TO-PRO3, we explored Gr1+ vs. CD11b+ cells populations (CD11b+Gr1low, CD11b+Gr1int, CD11b+Gr1high). Downstream analysis was performed based on CX3CR1+ and/or CD11b+ populations, which were gated from the “Live cells” gate. Figure S3. Flow cytometry analysis of myeloid cells in Gla+/- and control DRG cell suspensions from 20 - 24 weeks old female mice. (A) Representative scatter plots and histograms of CD11b and Gr1. (B) TLR4 expression and (C) higher CD163 expression in CX3CR1+ and/or Cd11b+ in GlaKO. (D) Dimensional reduction using tSNE and clustering analysis from each identified cell population in (A), (B) and (C). Pooled samples from 3 mice. 20 - 26 weeks wt female samples n=3, Gla+/- female samples n=4.

Additional file 1: Figure S1. Response of wild type T cells to PKD inhibitors in vitro. a, b MACS-sorted CD4+ T cells were treated with several concentrations of either one of two low molecular weight inhibitors (CRT0066101 in orange or CID2011756 in blue) one hour prior to stimulation with anti-CD3/CD28 antibodies. On day 2 of culture CD25 and Ki-67 expression were analyzed by flow cytometry to assess activation and proliferation, respectively. c IL-2 expression upon 4-hour stimulation of splenocytes with phorbol ester/ionomycin in the presence of brefeldin A and the depicted concentration of CRT was analyzed by intracellular staining and flow cytometry. Representative FACS histograms showing IL-2 (gated on CD4+ T cells) and summarizing graphs are shown. ≥ 2 independent experiments; n ≥ 4 mice analyzed individually.

Memory formation is a hallmark of T cell-mediated immunity, but how differentiation into either short-lived effector cells (SLECs, CD127(-)KLRG1(+)) or memory precursors cells (MPECs, CD127(+)KLRG1(-)) and subsequent regulation of long-term memory is adjusted is incompletely understood. Here, we show that loss of the nuclear orphan receptor NR2F6 in germ-line Nr2f6-deficient mice enhances antigen-specific CD8(+) memory formation up to 70 days after bacterial infection with Listeria monocytogenes (LmOVA) and boosts inflammatory IFN-gamma, TNF alpha, and IL-2 cytokine recall responses. Adoptive transfer experiments using Nr2f6(-/-) OT-I T-cells showed that the augmented memory formation is CD8(+) T-cell intrinsic. Although the relative difference between the Nr2f6(+/+) and Nr2f6(-/-) OT-I memory compartment declines over time, Nr2f6-deficient OT-I memory T cells mount significantly enhanced IFN-gamma responses upon reinfection with increased clonal expansion and improved host antigen-specific CD8(+) T-cell responses. Following a secondary adoptive transfer into naive congenic mice, Nr2f6-deficient OT-I memory T cells are superior in clearing LmOVA infection. Finally, we show that the commitment to enhanced memory within Nr2f6-deficient OT-I T cells is established in the early phases of the antibacterial immune response and is IFN-gamma mediated. IFN-gamma blocking normalized MPEC formation of Nr2f6-deficient OT-I T cells. Thus, deletion or pharmacological inhibition of NR2F6 in antigen-specific CD8(+) T cells may have therapeutic potential for enhancing early IFN-gamma production and consequently the functionality of memory CD8(+) T cells in vivo.

Upon activation by antigen, B cells form germinal centres where they clonally expand and introduce affinity‐enhancing mutations into their B‐cell receptor genes. Somatic mutagenesis and class switch recombination (CSR) in germinal centre B cells are initiated by the activation‐induced cytidine deaminase (AID). Upon germinal centre exit, B cells differentiate into antibody‐secreting plasma cells. Germinal centre maintenance and terminal fate choice require transcriptional reprogramming that associates with a substantial reconfiguration of DNA methylation patterns. Here we examine the role of ten‐eleven‐translocation (TET) proteins, enzymes that facilitate DNA demethylation and promote a permissive chromatin state by oxidizing 5‐methylcytosine, in antibody‐mediated immunity. Using a conditional gene ablation strategy, we show that TET2 and TET3 guide the transition of germinal centre B cells to antibody‐secreting plasma cells. Optimal AID expression requires TET function, and TET2 and TET3 double‐deficient germinal centre B cells show defects in CSR. However, TET2/TET3 double‐deficiency does not prevent the generation and selection of high‐affinity germinal centre B cells. Rather, combined TET2 and TET3 loss‐of‐function in germinal centre B cells favours C‐to‐T and G‐to‐A transition mutagenesis, a finding that may be of significance for understanding the aetiology of B‐cell lymphomas evolving in conditions of reduced TET function., TET (ten‐eleven‐translocation) enzymes promote gene expression by catalyzing the oxidation of 5‐methylcytosine in DNA. TET deficiency has been linked to defects in embryonic development and enhanced risk of myeloid malignancies, including B‐cell lymphomas. Here, Verena Labi and colleagues show using a conditional gene ablation strategy that TET function is vital for humoral immunity in vivo. TET activity guides the transition of germinal center B cells to antibody‐secreting plasma cells and promotes antibody isotype switching (class‐switch recombination). Loss of TET function favors C‐to‐T and G‐to‐A mutagenesis during somatic hypermutation, a finding of potential significance for understanding the etiology of B‐cell lymphomas.