SummaryB lymphocytes can suppress immunity through interleukin (IL)-10 production in infectious, autoimmune, and malignant diseases. Here, we have identified a natural plasma cell subset that distinctively expresses the inhibitory receptor LAG-3 and mediates this function in vivo. These plasma cells also express the inhibitory receptors CD200, PD-L1, and PD-L2. They develop from various B cell subsets in a B cell receptor (BCR)-dependent manner independently of microbiota in naive mice. After challenge they upregulate IL-10 expression via a Toll-like receptor-driven mechanism within hours and without proliferating. This function is associated with a unique transcriptome and epigenome, including the lowest amount of DNA methylation at the Il10 locus compared to other B cell subsets. Their augmented accumulation in naive mutant mice with increased BCR signaling correlates with the inhibition of memory T cell formation and vaccine efficacy after challenge. These natural regulatory plasma cells may be of broad relevance for disease intervention.

Synthetic oligodeoxyribonucleotides (ODNs) containing CpG (unmethylated deoxycytidylyl-deoxyguanosine dinucleotide) motifs activate endosomal TLR9. The nucleotide sequence, length, and dimerization properties of ODNs modulate their activation of TLR9. We performed a systematic investigation of the sequence motifs of B-class and C-class phosphodiester ODNs to identify the sequence properties that govern TLR9 activation. ODNs shorter than 21 nt and with the adenosine adjacent to the cytidine-guanosine (CG) dinucleotide motif led to a significant loss of the propensity to activate TLR9. The distance between the stimulatory CpG motifs within the ODN fine-tunes the activation of B cells. The minimal ODNs that activate human TLR9 comprise 2 CG dinucleotides separated by 6–10 nt, where the first CpG motif is preceded by the 5′-thymidine and the elongated poly-thymidine tail at the 3′ end of the ODN. The minimal sequence provides insight into the molecular mechanism of TLR9 ligand recognition. On the basis of sequence requirements, we conclude that two binding sites with different affinities for CG are formed in the human TLR9 dimer, with a very stringent binding site interacting with the 5′ CpG motif.

Synthetic oligodeoxyribonucleotides (ODNs) containing unmethylated CpG recapitulate the activation of TLR9 by microbial DNA. ODNs are potent stimulators of the immune response in cells expressing TLR9. Despite extensive use of mice as experimental animals in basic and applied immunological research, the key sequence determinants that govern the activation of mouse TLR9 by ODNs have not been well defined. We performed a systematic investigation of the sequence motif of B class phosphodiester ODNs to identify the sequence properties that govern mouse TLR9 activation. In contrast to ODNs activating human TLR9, where the minimal sequence motif for the receptor activation comprises a pair of closely positioned CpGs we found that the mouse TLR9 requires a single CpG positioned 4–6 nt from the 5′-end. Activation is augmented by a 5′TCC sequence one to three nucleotides from the CG. The distance of the CG dinucleotide of four to six nucleotides from the 5′-end and the ODN’s length fine-tunes activation of mouse macrophages. Length of the ODN <23 and >29 nt decreases activation of dendritic cells. The ODNs with minimal sequence induce Th1-type cytokine synthesis in dendritic cells and confirm the expression of cell surface markers in B cells. Identification of the minimal sequence provides an insight into the sequence selectivity of mouse TLR9 and points to the differences in the receptor selectivity between species probably as a result of differences in the receptor binding sites.

Background: UNC93B1 sorts nucleic acid-sensing TLRs to endosomes. Results: Stimulation of HUVEC by TLR3, but not other TLR agonists, up-regulates the endogenous UNC93B1, which translocates TLR3 to plasma membrane. Poly(I:C) strongly potentiates activation of TLR9 via UNC93B1. Conclusion: Up-regulation of UNC93B1 through TLR3 activation forms a positive feedback loop that primes cells for the augmented response against infection. Significance: This work presents unique TLR3-UNC93B1 interrelationship.

We show that substrate specificity of Toll-like receptor 3 (TLR3) is due to the presence of two binding sites in the ectodomain, separated by 50 A. This corresponds to two turns of a double-stranded RNA duplex, allowing differentiation between nucleic acids in the A- or B-type conformation. We propose that there are different arrangements of TLR3 ectodomains along the double-stranded RNA that could modulate the strength of the interferon response.

Triple negative breast cancer, characterised by the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (HER-2), has a poor prognosis mostly due to increased rate of distant metastases 1,2 . During the process of metastasation, cancer cells in primary tumour locally invade the tumour-associated stroma, detach from the invasion front of the tumour, and enter the lymphatic and/or blood vessels. Circulating cancer cells ultimately migrate through the capillary wall in distant tissues, re-attach to the extracellular matrix, and proliferate in a new microenvironment 3 . Once cancer cells detach from the main tumour mass, they must resist anoikis, a programmed cell death induced by extracellular matrix detachment 4 . MDA-MB-231 cells, the most commonly used in vitro model of triple negative breast cancer 5 , are highly metastatic and tumorigenic 5 . They form colonies in an anchorage-independent condition 6 , and are resistant to anoikis 7 . Albeit breast cancer cells must detach from extracellular matrix in order to metastasise in vivo [8][9][10][11] , floating MDA-MB-231 cells in vitro are commonly thought to be dead. Only a few studies investigated the viability of floating MDA-MB-231 cells in vitro [12][13][14] . Metabolic adaptations enable survival of cancer cells in an anchorage-independent condition 6,[15][16][17][18] . By stimulating glucose uptake, oncogenes restore redox and energy balance and prevent anoikis in breast cancer cells 18 .

TLR9 acts as a first-line host defense against pathogens recognizing DNA comprising unmethylated CpG motifs present in bacteria and viruses. Species- and sequence-specific recognition differences were demonstrated for TLR9 receptors. Activation of human (h)TLR9 requires a pair of closely positioned CpG motifs within oligodeoxyribonucleotides (ODNs), whereas mouse TLR9 is effectively activated by an ODN with a single CpG motif. Molecular model-directed mutagenesis identified two regions, site A and site B, as important for receptor activation. Amino acid residues Gln and Arg within site A contribute to the sequence-specific recognition by hTLR9 in determining the bias for two appropriately spaced CpG motifs within immunostimulatory ODNs. Mutation of Gln at site B, in combination with Gln and Arg mutations of mouse counterparts, increased activation of hTLR9 by mouse-specific ODN, mammalian genomic DNA, and bacterial DNA. We propose that the double CpG motif sequence-specificity of hTLR9 results in decreased activation by ODNs with a lower frequency of CpG motifs, such as from mammalian genomic DNA, which increases hTLR9 selectivity for pathogen versus host DNA.

Toll-like receptors encounter a diversity of degradation products in endosomes. TLR7 and TLR8 have been shown to be activated by RNA degradation products. Here we show that although TLR9 requires single-stranded DNA longer than 20 nucleotides for a robust response, TLR9 activation is augmented by CpG-containing oligodeoxyribonucleotides (sODNs) as short as 2 nucleotides, which, by themselves, do not induce activation in cell cultures, as well as in mice. sODNs also activate human TLR9 in combination with ODNs containing a single CpG motif that by themselves do not activate human TLR9. The specific sequence motif of sODN and colocalization of ODN and sODN suggest that the mechanism of activation involves binding of both ODN and sODN to TLR9. sODNs augment TLR9 activation by mammalian genomic DNA indicating the role of short DNA degradation products in the endosomes in response to infection or in autoimmune disease, particularly at limiting concentrations of ODNs.