Project description:B cells potentially play a role in the immune response to melanoma, including during treatment with immune modulators. We profiled effects of 3M-052 on gene expression in draining lymph nodes of syngeneic mice bearing B16 melanoma tumors. When B16 tumors reached 30-40 mm2 in size, they were injected intratumorally once with 3M-052 or vehicle control.
Project description:Ewings Sarcoma (ES) belongs to the group of bone cancers defined by the existence of a certain EWS-ETS fusion gene. In this study we use the model cell line CADO-ES1 (EWSR1-ERG fusion gene) to characterize the tumor biology of a versatile ES side-population (SP). We aim to compare SP- and non-SP-cells to identify specific characteristics of the SP which points towards a tumor driving functionality of the SP. Due to some stem cell like properties of the SP fraction a comparison to MSCs and normal fibroblasts as a control are also performed.
Project description:Authorization of the Matrix-M-adjuvanted R21 vaccine by three countries and its subsequent endorsement by the World Health Organization (WHO) for malaria prevention in children is a milestone in the fight against malaria. Yet, to meet the unprecedented demand for malarial vaccines, there is a pressing need for additional adjuvants that induce robust and durable vaccine-induced immunity. Here, we performed a comparative assessment of three clinically relevant adjuvants (an alum formulation of the TLR7/8 agonist 3M-052 (3M-052+Alum), the TLR4 agonist GLA-LSQ (GLA in liposome QS-21 formulation), and Matrix-M, the currently approved adjuvant for R21) for their capacity to induce durable immune responses to the R21 malaria vaccine in non-human primates. Immunization of macaques with R21 adjuvanted with 3M-052+Alum on a 0, 8, and 24-week schedule elicited anti-circumsporozoite antibody responses comparable in magnitude to the R21/Matrix-M vaccine and persisted up to 72 weeks with a half-life of 337 (264 – 459) days. A booster dose at 72 weeks induced an antigen-specific recall response, similar to the R21/Matrix-M vaccination. In contrast, R21/GLA-LSQ immunization induced a considerably lower and short-lived response. Consistent with the durability of serum antibody responses, Matrix-M and 3M-052+Alum induced long-lived plasma cells in the bone marrow and other tissues, including the spleen, but GLA-LSQ stimulated only short-lived plasmablasts. Finally, we show distinct innate immune signatures early after vaccination with these adjuvants. While 3M-052+Alum stimulated potent and persistent antiviral transcriptional and cytokine signatures after primary and booster immunizations, Matrix-M induced an enhanced expression of interferon- and Th2-related signatures more highly after the booster vaccination. Collectively, these findings provide a comparative database on the immune responses of three clinically relevant adjuvants with R21 and highlight the promise of 3M-052+Alum as an additional adjuvant for the R21 malaria vaccine.
Project description:Class-switching to IgG2a/c in mice is a hallmark response to intracellular pathogens. T cells can promote class-switching and the predominant pathway for induction of IgG2a/c antibody responses has been suggested to be via stimulation from Th1 cells. We previously formulated CAF®01 (cationic liposomes containing dimethyldioctadecylammonium bromide (DDA) and Trehalose-6,6-dibehenate (TDB)) with the lipidated TLR7/8 agonist 3M-052 (DDA/TDB/3M-052), which promoted robust Th1 immunity in newborn mice. When testing this adjuvant in adult mice using the recombinant Chlamydia trachomatis (C.t.) vaccine antigen CTH522, it similarly enhanced IgG2a/c responses compared to DDA/TDB, but surprisingly reduced the magnitude of the IFN-g+ Th1 response in a TLR7 agonist dose-dependent manner. Single cell RNA-sequencing revealed that DDA/TDB/3M-052 liposomes initiated early transcription of class-switch regulating genes directly in pre-germinal center B cells. Mixed bone marrow chimeras further demonstrated that this adjuvant did not require Th1 cells for IgG2a/c switching, but rather facilitated TLR7-dependent T-bet programming directly in B cells. This study underlines that adjuvant-directed IgG2a/c class-switching in vivo can occur in the absence of T cell help, via direct activation of TLR7 on B cells and positions DDA/TDB/3M-052 as a powerful adjuvant capable of eliciting type I-like immunity in B cells without strong induction of Th1 responses.
Project description:JmjC domain containing protein 14 (JMJ14) is an H3K4-specific histone demethylase that plays important roles in RNA-mediated gene silencing and flowering time regulation in Arabidopsis. However, how JMJ14 is recruited to their target genes remains unclear. Here, we show that the C-terminal FYRN and FYRC domains of JMJ14 are required for RNA silencing and flowering time regulation. Chromatin binding of JMJ14 is lost upon deletion of its FYRN and FYRC domains, along with increased H3K4me3. FYRN and FYRC domains interact with a pair of NAC domain containing transcription factors, NAC050 and NAC052. Genome-wide analysis revealed that JMJ14 and NAC050/052 share a set of common target genes with CTTGNNNNNCAAG consensus sequences. Mutations in either NAC052 or NAC050 impair RNA-mediated gene silencing. Thus, our findings demonstrate an important role of FYRN and FYRC in targeting JMJ14 through direct interaction with NAC050/052 transcription factors, which reveals a novel mechanism of recruiting a histone demethylases to its target loci in higher plants. anti-HA ChIP-seq were performed with six samples: Col, NAC050-HA, NAC052-HA, jmj14-1, JMJ14-HA and JMJ14-FYR-HA. Anti-H3K4me3 ChIP were performed with four samples: Col, jmj14-1, JMJ14-HA and JMJ14-FYR-HA. (NAC050-HA indicates PNAC050::NAC050-HA nac050-1, NAC052-HA indicates PNAC052::NAC052-HA nac052-2, JMJ14-HA indicates PJMJ14::JMJ14-HA jmj14-1, JMJ14-FYR-HA indicates PJMJ14::JMJ14-FYR-HA jmj14-1)