Purpose: Murine monocytes require A-type paired immunoglobulin-like receptors (PIR-A) to specifically recognize and acquire memory to major histocompatibility complex I antigens. Beyond the need for PIR-A, little is known about the mechanisms of specific innate immune memory. In this study, we aim to explore possible mechanisms of monocyte memory by investigating the epigenetic and transcriptional changes that specifically occur after allogeneic stimulation in splenic monocytes.
Methods: B6.RAG-/- γc-/- (BRG) mice were immunized by intraperitoneal injection of 20 million Balb/c irradiated splenocytes. Splenic monocytes were FACS sorted at 0-, 3-, 7- and 28-days post-immunization. Simultaneous snRNAseq and snATACseq was then performed. To identify the changes that are specific to allo-stimulation and related to monocyte memory, monocytes were also sorted and sequenced from BRG mice injected with irradiated B6 splenocytes and allo-immunized BRG PIR-A-/- mice at day 7 after injection. PBMCs were collected from 12 transplant patients with or without rejection. ScRNAseq was performed on the PBMCs after depletion of T and B cells.
Results: Weighted nearest neighbor UMAP represented 4 visually distinct cell neighborhoods (N). N1 and N2 increased in abundance after allo-stimulation. Differential gene expression analysis revealed that N1 highly expressed genes encoding cell cycle proteins, Ly-6C, and PIR-A. Clustering resulted in the division of N2 into 3 clusters (C1-C3). Flow cytometric analysis which included EDU pulse-chase confirmed the patterns seen in the sequencing data. Pseudotime multiomic trajectory inference using Multivelo revealed an influx of cells from N1 to N2 at D7. Real-time multiomic trajectory inference using Waddington OT supported that N1 is the starting state for memory formation in response to allogeneic non-self. Real time and pseudotime trajectory inference suggested that the differentiation pathway connects N1 to N2 then splits to N3 and N4. Static and dynamic gene regulatory network inference using Dictys revealed key transcription factors in N1 for monocyte memory formation and response. Similar findings were uncovered in the transcriptomic landscape of monocytes from transplant patients’ PBMCs.
Conclusion: Specific Innate Immune Memory is sourced from a monocyte progenitor subset specifically responding to allo-stimulation with unique transcriptional and epigenetic changes which then pass down to its progeny.