Project description:To understand the molecular determinants of B12(VHH)-CAR-T polyfunctionality, we simultaneously measured the protein and RNA expression levels of 95 single CD4+ B12(VHH)-CAR-T cells isolated from IMR5 tumor-bearing mouse and re-stimulated with IMR5 tumor cells in vitro. We identified two CAR-T clusters, low polyfunctionality and high polyfunctionality subsets, in a 2D t-SNE plot. Moreover, our single-cell mRNA expression profiling revealed 32 genes that displayed statistically significant, concordant differences between the two cell subsets.
Project description:Camelids are capable of producing both conventional tetrameric antibodies (Abs) and dimeric heavy-chain antibodies (HCAbs). While B cells generating these two types of Abs exhibit distinct B-cell receptors (BCRs), it remains unclear whether these two B cell populations differ in their phenotypes and developmental processes. Here, we collected eight PBMC samples before and after immunization from four Bactrian camels and conducted single-cell 5’ RNA sequencing. We characterized the functional subtypes and differentiation trajectories of circulating B cells in camels, including native B cells, memory B cells, intermediate B cells, atypical B cells, and plasma cells. Additionally, we reconstructed single-cell BCR sequences and revealed the IGHV and IGHC gene types. We found that B cells with variable genes of HACbs (VHH) were widely present in various functional subtypes and showed highly overlapping differentiation trajectories to B cells with variable genes of conventional Abs (VH). After immunization, the transcriptional changes in VHH+ and VH+ B cells were also largely consistent. Our study not only elucidates the cellular context of HCAb production in camels, but also lays the foundation for the development of single B cell-based nanobody screening.
Project description:Additive effects of TNF and IL-6 were evaluated in cell-based model for rheumatoid arthritis (RA) and then compared to treatment with anti-TNF/IL-6 NANOBODY® VHH, Humira and Sylvant by RNA-seq
Project description:The growing appreciation of immune cell-cell interactions within disease environments has led to significant efforts to develop highly effective protein- and cell-based immunotherapies. However, characterizing these complex cell-cell interfaces in high resolution remains challenging. Thus, technologies that can leverage therapeutic-based modalities to profile intercellular environments offer the opportunity to study cell-cell interactions with molecular-level insight. To address this, we introduce Photocatalytic Cell Tagging (PhoTag), a platform for profiling cell-cell interactions utilizing a single domain antibody (VHH) conjugated to a photoactivatable flavin-based cofactor. Upon irradiation with visible light, the tethered flavin photocatalyst generates phenoxy radical tags for targeted labeling within cell-cell contact regions. Using anti-PD-1 or anti-PD-L1 VHH flavin conjugates, we demonstrate that PhoTag achieves highly selective synaptic labeling in antigen presenting cell-T cell co-culture systems. By combining the high resolution transcellular biotinylation capability of PhoTag with multi-omics single cell sequencing, we interrogated transient interactions between peripheral blood mononuclear cell (PBMC) populations and Raji PD-L1 B cells and discovered that specific T cell subtypes can transiently interact more efficiently than others. We envision that the spatiotemporal and modular nature of PhoTag will enable its broad utilization for detailed profiling of intercellular interactions across different biological systems.
