TLR-9 has a transmembrane domain and other features of a type 1 membrane protein.

TLR9 is ER protein. DNA is internalized into early endosmome. TLR translocates to early endosome where it binds the DNA and becomes activated. When TLR binds DNA is undergoes conformation change.

The physiologic ligands for TLR9 are bacterial dsDNA fragments with unmethylated cyotidine phosphate guanosine (CpG) dinucleotides. (US 2023/0404642).

Bacterial DNA is unmethylated and contains CpG motifs. This is how the immune response differentiates from host and bacterial DNA. Immune activation by CpG-B ODN depends on TLR-9, as mice genetically deficient in this molecule show no CpG induced activation of B cells, DC or NK cells. Unmethylated CpG motifs are prevalent in bacterial but not vertebrate genomic DNAs. About 70% of the cytosines in vertebrate DNA are methylated. Oligodeoxynucleotides (ODN) containing CpG motifs activate host defense mechanisms leading to innate and acquired immune resposnes. Optimal CpG motifs for activating mouse or rabbit immune cells have the general formula, purine-purine-CG-pyrimidine-pyrimidine. The best CpG reported was GACGTT. For activating human cells, the optimal motif reported is GTCGTT. The immune stimulatory effects of the ODN are enhanced if the ODN has a TpC dinucleotide on the 5′ end and is pyrimidine rich on the 3′ side. Immune stimulatory properties of a particular ODN are also affected by the number and spacing of the CpG motifs, the presence of poly G sequences or other flanking sequences in the ODN and the ODN backbone. DNA vaccines typically contain several hundred CpG motifs, some of which are in an immunostimulatory context, and others of which are CpG-N motifs. Addition of just two immune stimulatory CpG motifs to a DNA vaccine can enhance its ability to induce an antigen-specific immune response.

Following their uptake into lymphocytes, ODN appear to be located within the endosomal compartment. Chloroquine and bafilomycin A, which interfere with endosomal acidification and/or maturation, completely block the immune stimulatory activity of CpG DNA at low concentrations. These compounds must act at a very early step in the CpGp induced signaling pathway because their inhibitory effects are apparent by 5 min and they block all of the signaling pathways known to be induced by CpG. The TLR9 protein determine the species specificity of CpG motifs. Circumstantial evidence suggests that TLR9 may be present in the endosomes, where it could interact with CpG DNA.

The recognition of CpG motifs requires TLR9, which triggers alterations in cellular redox balance and the induction of cell signaling pathways including MAPKs and NFkB. CpG DNA has potent transcription activating effects on macrophages and DC, which leads to the increased transcription of TNFalpha, IL-1beta, plasminogen activator 2, IL-6, IL-12, Type I interferons and several costimulatory and antigen presenting molecules such as class II MHC, CD80, CD86, CCR7 and CD40. Of interest, CpG also activates the ERK pathway in primary macrophages and RAW264.7, a macrophage like cell line, and this contributes to CpG induced TNF production but has a negative feedback effect on the IL-12 p40 promoter which results in decreased release of IL-12.

In addition to secreting cytokines and Ig, CpG-activated B cells express increased levels of the Fcy receptor and costimulatory molecules such as class II MHC, CD80 and CD86.

Among human DC subsets, so far only the pDC are clearly demonstrated to be directly activated by CpG DNA, which induces them to have growth factor independent survival in culture, resistance to IL-4 induced apoptosis, increased surface expression of MHC class II, ICAM-1 and the costimulatory molecules CD40, CD54, CD80 and CD86, cytokine seretion (IL-6, TNFalpha, IFNalpha secretion, chemokine production (IL-8, IP-10, GM-CSF) and maturation to become CD83 bright with increased activation of allogeneic T cells.

TLR9 is an intracellular pattern recognition receptor present in the endosomal compartment of plasmacytoid dendritic cells (DCs), macrophages, natural killer cells and other APCs in mice. In human TLR9 is primarily expressed in B cells and pDCs. (US 20230404642)

TLR9 agonsists are artificial ogligonucleotdies bearing unmethylated CpG motifs. They can induce a signaling cascade that leads to transcriptional programs that result in inflammatory processes, enhanced killing of cancer cells as well as in the generation of adaptive immune resposnes. Data suggest that intatumoral adminsitraiton of TLR9 agonsits improved APC activaiton, in particualr pDCs in TDLNs resulting in proinflammtory IL-12 and Type I INF genes. IFNalpha has direct effects on tumors, including inhibition of angiogenesis, antiproliferative effects adn incdrease in MHC- expression by tumor cells, which ehances targt visibility and facilitates killing of tumor cells by T cells. IFNalpah enhaces NK killing ability and maturation of covnention DCs. Additionallyk TLR9 agonsits can icnreased epxression of co-stimulatory molecuels CD80 and CD86, increase in IP10 (CXCL10) a chemoattractant of monocytes, macrophages, DCs, T cells, NK cells and incdreaes expressio of the lymph node homing signal CCD7 which ultimately resutls in increased T cell priming and tumor rejection, and in some instances, potent adscopal effects in dsital treated tumors. (US 2023/0404642)