Journal: bioRxiv
Article Title: Defining the Immunomodulatory Determinants of 3-mer Oligonucleotides on TLR7 and TLR8 sensing
doi: 10.64898/2026.04.29.720527
Figure Lengend Snippet: ( a ) Bone-marrow-derived macrophages (BMDMs) from Tlr7 Y264H mice were treated for 24 h with 1 μM of BMS905 and 5 μM of GUC-v16 oligo prior to RNA purification for RNA-sequencing or RT-qPCR analyses. RT-qPCR analyses of Fpr1, Slc13a3, Cd300e, Itgal, Nfkbiz, Slamf9, Clec4a2 and Clec4a1 reported to 18S in RNA lysates from primary BMDMs from 3 independent Tlr7 Y264H mice. Data are shown relative to non-treated (NT) mice (± s.e.m. and two-way ANOVA with uncorrected Fisher’s LSD tests shown compared to NT Tlr7 Y264H condition). ( b ) WT C57/BL6 mice were injected i.v. with 200 μg of GUC-v16 conjugated with in vivo -jetPEI® or vehicle (glucose solution) for 1 h prior to i.p. injection of 25 μg R848 for 2 h before collection of spleens. RT-qPCR analyses of Fpr1, Fpr2, Marco, Nfkbiz and Tnf reported to Gapdh , from spleen lysates; data are shown relative to non-treated (NT) mice (± s.e.m. and two-way ANOVA with uncorrected Fisher’s LSD tests shown compared to R848 mice). Each dot represents an individual mouse with bars showing the mean of n=5 mice/group is shown (± s.e.m. and one-way ANOVA with uncorrected Fisher’s LSD tests shown compared to R848 group). ( c ) Aldara cream was applied topically to the back of WT C57/BL6 mice directly following, or not, application of 100 μl of highly pure 2.5% GUC-v16 cream oligonucleotide (>99.4%). After four days, mice were humanely euthanised and back skin collected, and lysed for RNA purification. RT-qPCR analyses of indicated genes reported to that of 18S expression, relative to NT mice. Data are representative of 2 independent experiments. Mean of n=3 NT and n=8 Aldara/Aldara+GUC-v16 mice/group is shown (± s.e.m. and one-way ANOVA with uncorrected Fisher’s LSD tests shown compared to Aldara group). ( d-f ) Wild type C57BL/6J mice were treated i.t. with 2.5 μg of naked GUC-v16 in water for 1 h prior to. i.t. injection of 50 μg of R848. BALF ( d, f ) were harvested 7 h post R848 injection and analysed by cytospin differential cell counting for total cell counts ( d ) and using an MSD multiplex assay for cytokine quantification ( f ). Cytokine levels in plasma were also quantified using an MSD multiplex assay ( e ). Data shown are from 1 experiment, representative of n=2 independent experiments. Mean of n=5 NT and n=7 R848/R848+GUC-v16 mice/group is shown (± s.e.m. and one-way ANOVA with uncorrected Fisher’s LSD tests shown compared to NT [d] or R848 [ e,f ] group). Whole blood from 4 ( g ) or 1 ( h ) healthy controls or patients with UNC93B1 E92G mutation ( h ) was pre-treated with indicated 3-mer concentration for 30 min prior to stimulation with indicated amount of R837 or 50ng/ml TL8-506 for 24 h prior to cytokine bead analyses. ( g ) For each control, cytokine levels were averaged for each technical replicate (6/sample), background corrected to unstimulated samples only, and reported to cytokine levels from stimulation only controls. Heat maps were generated using scale from 0-1 (see methods), were 1 is the cytokine level of the agonist only control. ( g-h ) Data shown are from a minimum of two independent experiments – conducted on two independent days. ( i ) HEK-TLR7 cells were pre-treated with indicated concentration of oligo for 1 h prior to overnight stimulation with 10% of synovial fluid from RA patient. SEAP absorbances were background-corrected using the non-treated condition, and are shown as relative expression to the synovial fluid only control; Neg. is a non-oligo condition used as control. Data shown are averaged from synovial fluid stimulations from n=8 patients (± s.e.m. and two-way ANOVA with uncorrected Fisher’s LSD tests shown compared to Neg condition).
Article Snippet: HEK-BlueTM TLR7 (Invivogen, hkb-htlr7v2) reporter cells were cultured according to the manufacturer’s instructions and plated in complete DMEM prior to treatment.
Techniques: Derivative Assay, Purification, RNA Sequencing, Quantitative RT-PCR, Injection, In Vivo, Cream, Expressing, Cell Counting, Multiplex Assay, Clinical Proteomics, Mutagenesis, Concentration Assay, Control, Generated
InvivoGen is a verified supplier 
InvivoGen manufactures this product 
![( a , e and h ) <t>HEK-TLR7</t> and HEK-TLR8 cells were pre-treated ∼30 min with 125 nM (TLR7) and 5 μM (TLR8) of the indicated oligos prior to overnight stimulation with 1 μg/ml of R848 followed by luciferase assay. Data are mean of n=3 independent experiments. Data were background-corrected using the non-treated (NT) condition and are shown as relative expression to R848 only (± s.e.m. and one-way ANOVA with uncorrected Fisher’s LSD tests shown compared to GUC-v1+R848 [ a (TLR7)], GUC+R848 [ a (TLR8), and e ] and R848 [ h ] condition; a [TLR7 and TLR8]: P<0.0001 ; e [TLR7]: P<0.0001 and [TLR8]: P=0.0005 ; h [TLR7 and TLR8]: P<0.0001 ). ( c , g and j ) HEK-TLR7 cells were pre-treated ∼30 min with 50 nM of the indicated oligos prior to overnight stimulation with 1 μg/ml of R848 followed by luciferase assay. Data are mean of n=3 independent experiments. Data were background-corrected using the non-treated (NT) condition and are shown as relative expression to R848 only (± s.e.m. and one-way ANOVA with uncorrected Fisher’s LSD tests shown compared to GUC+R848 ( c and g ) and GUC-v1+R848 ( j ) condition; c , g and j : P<0.0001 ). ( d ) HEK-TLR7 cells were pre-treated ∼30 min with different doses (1000 nM, 500 nM, 250 nM, 125 nM and 62.5 nM) of the indicated oligos and Enpatoran (100 nM, 50 nM, 25 nM, 12.5 nM and 6.25 nM) prior to overnight stimulation with 1 μg/ml of R848 followed by luciferase assay. Data are mean of n=3 independent experiments. Data were background-corrected using the non-treated (NT) condition and are shown as relative expression to R848 only (± s.e.m. and one-way ANOVA with uncorrected Fisher’s LSD tests shown compared to R848 only condition).](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_27/10__64898_slash_2026__04__29__720527/10__64898_slash_2026__04__29__720527___F2.large.jpg)
![( a ) Bone-marrow-derived macrophages (BMDMs) from Tlr7 Y264H mice were treated for 24 h with 1 μM of BMS905 and 5 μM of GUC-v16 oligo prior to RNA purification for RNA-sequencing or RT-qPCR analyses. RT-qPCR analyses of Fpr1, Slc13a3, Cd300e, Itgal, Nfkbiz, Slamf9, Clec4a2 and Clec4a1 reported to 18S in RNA lysates from primary BMDMs from 3 independent Tlr7 Y264H mice. Data are shown relative to non-treated (NT) mice (± s.e.m. and two-way ANOVA with uncorrected Fisher’s LSD tests shown compared to NT Tlr7 Y264H condition). ( b ) WT C57/BL6 mice were injected i.v. with 200 μg of GUC-v16 conjugated with in vivo -jetPEI® or vehicle (glucose solution) for 1 h prior to i.p. injection of 25 μg R848 for 2 h before collection of spleens. RT-qPCR analyses of Fpr1, Fpr2, Marco, Nfkbiz and Tnf reported to Gapdh , from spleen lysates; data are shown relative to non-treated (NT) mice (± s.e.m. and two-way ANOVA with uncorrected Fisher’s LSD tests shown compared to R848 mice). Each dot represents an individual mouse with bars showing the mean of n=5 mice/group is shown (± s.e.m. and one-way ANOVA with uncorrected Fisher’s LSD tests shown compared to R848 group). ( c ) Aldara cream was applied topically to the back of WT C57/BL6 mice directly following, or not, application of 100 μl of highly pure 2.5% GUC-v16 cream oligonucleotide (>99.4%). After four days, mice were humanely euthanised and back skin collected, and lysed for RNA purification. RT-qPCR analyses of indicated genes reported to that of 18S expression, relative to NT mice. Data are representative of 2 independent experiments. Mean of n=3 NT and n=8 Aldara/Aldara+GUC-v16 mice/group is shown (± s.e.m. and one-way ANOVA with uncorrected Fisher’s LSD tests shown compared to Aldara group). ( d-f ) Wild type C57BL/6J mice were treated i.t. with 2.5 μg of naked GUC-v16 in water for 1 h prior to. i.t. injection of 50 μg of R848. BALF ( d, f ) were harvested 7 h post R848 injection and analysed by cytospin differential cell counting for total cell counts ( d ) and using an MSD multiplex assay for cytokine quantification ( f ). Cytokine levels in plasma were also quantified using an MSD multiplex assay ( e ). Data shown are from 1 experiment, representative of n=2 independent experiments. Mean of n=5 NT and n=7 R848/R848+GUC-v16 mice/group is shown (± s.e.m. and one-way ANOVA with uncorrected Fisher’s LSD tests shown compared to NT [d] or R848 [ e,f ] group). Whole blood from 4 ( g ) or 1 ( h ) healthy controls or patients with UNC93B1 E92G mutation ( h ) was pre-treated with indicated 3-mer concentration for 30 min prior to stimulation with indicated amount of R837 or 50ng/ml TL8-506 for 24 h prior to cytokine bead analyses. ( g ) For each control, cytokine levels were averaged for each technical replicate (6/sample), background corrected to unstimulated samples only, and reported to cytokine levels from stimulation only controls. Heat maps were generated using scale from 0-1 (see methods), were 1 is the cytokine level of the agonist only control. ( g-h ) Data shown are from a minimum of two independent experiments – conducted on two independent days. ( i ) HEK-TLR7 cells were pre-treated with indicated concentration of oligo for 1 h prior to overnight stimulation with 10% of synovial fluid from RA patient. SEAP absorbances were background-corrected using the non-treated condition, and are shown as relative expression to the synovial fluid only control; Neg. is a non-oligo condition used as control. Data shown are averaged from synovial fluid stimulations from n=8 patients (± s.e.m. and two-way ANOVA with uncorrected Fisher’s LSD tests shown compared to Neg condition).](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_27/10__64898_slash_2026__04__29__720527/10__64898_slash_2026__04__29__720527___F4.large.jpg)