Co-adjuvanting DDA/TDB liposomes with a TLR7 agonist allows for IgG2a/c class-switching in the absence of Th1 cells

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. Mice were immunized on day 0 and day 42 subcutaneously (s.c.) at the base of the tail with 2 μg recombinant CTH522 antigen in a volume of 200 μl TRIS/trehalose buffer (isotonic, pH 7.4) per immunization. Adjuvant doses were as follows: DDA/TDB liposomes (dose 250 μg/50 μg (DDA/TDB)) and DDA/TDB/3M-052_M liposomes (dose of 250 μg/50 μg (DDA/TDB)) with incorporation of 3M-052 in doses of 2 μg. TRIS (Naive mice) received 200 μL TRIS buffer. The lymph node where investigated on day 44. For each vaccine group, cells from the Lymph node of three mice were pooled and run on the 10x Chromium (10x Genomics) following a library preparation with Chromium Next GEM Single cell 3’ Reagent Kit v.3.1 (10x Genomics). Libraries were sequenced on the NovaSeq 6000 with the NovaSeq 6000 SP reagent kit v1.5 (Illumina). BCL files were converted to FASTQ files with bcl2fastq (Illumina) and data were then processed through Cellranger (10x Genomics, v6.1.2) and aligned to the mm10 genome. The raw transcript count matrix generated from alignment was loaded into R (v4.3.0) using the Seurat (v.4.3.0) package. A Seurat object was created (min.cells=3, min.features=200). Data were filtered (nFeature_RNA > 300 & nFeature_RNA < 3000 & percent.mt < 5), normalized (NormalizeData function from Seurat) and variable features were identified (FindVariableFeatures, selection.method=vst). Doublets were filtered out using DoubletFinder (v2.0.3). The three samples were integrated (FindIntegrationAnchors, IntegrateData from Seurat), scaled (ScaleData) and dimension-reduced by PCA (RunPCA). The first 13 PCs were used to construct a SNN network and a graph-based clustering approach. Louvain algorithm, was applied to identify cell clusters with the resolution set to 0.7. UMAP was constructed (RunUMAP), and differentially expressed markers for each cluster were found (FindAllMarkers, min.pct=0.25, logfc.threshold=0.25, test.use = wilcox). The three B cells clusters were subset and re-clustered (normalized, variable features was identified, integrated, scaled and PCA transformed). The first 13 PCs were used to construct the UMAP (resolution=0.5).