Universal recording of cell-cell contacts in vivo for interaction-based transcriptomics

Abstract

Cellular interactions are essential for tissue organization and functionality. In particular, immune cells rely on direct and usually transient interactions with other immune and non-immune populations to specify and regulate their function. To study these "kiss-and-run" interactions directly in vivo, we previously developed LIPSTIC (Labeling Immune Partnerships by SorTagging Intercellular Contacts), an approach that uses enzymatic transfer of a labeled substrate between the molecular partners CD40L and CD40 to label interacting cells. Reliance on this pathway limited the use of LIPSTIC to measuring interactions between CD4⁺ helper T cells and antigen presenting cells, however. Here, we report the development of a universal version of LIPSTIC (uLIPSTIC), which can record physical interactions both among immune cells and between immune and non-immune populations irrespective of the receptors and ligands involved. We show that uLIPSTIC can be used, among other things, to monitor the priming of CD8⁺ T cells by dendritic cells, reveal the cellular partners of regulatory T cells in steady state, and identify germinal center (GC)-resident T follicular helper (Tfh) cells based on their ability to interact cognately with GC B cells. By coupling uLIPSTIC with single-cell transcriptomics, we build a catalog of the immune populations that physically interact with intestinal epithelial cells (IECs) and find evidence of stepwise acquisition of the ability to interact with IECs as CD4⁺ T cells adapt to residence in the intestinal tissue. Thus, uLIPSTIC provides a broadly useful technology for measuring and understanding cell-cell interactions across multiple biological systems.

Figure 1.. The uLIPSTIC system.

(A, B) Schematic comparison of the original and universal LIPSTIC systems. In the original system (A), SrtA and G5 were brought into proximity by fusion to a receptor–ligand pair involved in a cell–cell interaction, allowing intercellular transfer of labeled substrate (LPETG) from donor cell “D” to acceptor cell “A.” In uLIPSTIC (B), SrtA and G5 (fused to the irrelevant protein Thy1.1) are anchored non-specifically to the cell membrane at high density; the enzymatic reaction is allowed to proceed when apposing membranes come within a short distance (< 14 nm) of each other, which can be driven by interactions between any receptor–ligand pair of the appropriate dimensions. (C) Computational model depicting the inter-membrane span of fully extended mSrtA upon transfer of the LPETG substrate onto G5-Thy1.1. (D,E) Populations of 293T cells co-transfected with high or low levels of either mSrtA or G5-Thy1.1 were co-incubated in the presence of biotin-LPETG for 30 min and analyzed by flow cytometry. (F) Rosa26^uLIPSTIC allele. Using the Ai9 high-expression backbone, a LoxP-flanked G5-Thy1.1 is followed by mSrtA. Cre-recombinase switches cells from “acceptor” (G5-Thy1.1⁺) to “donor” (mSrtA⁺) modes. (G) Rosa26^uLIPSTIC/+.CD4-Cre OT-II T cells were co-cultured with Rosa26^uLIPSTIC/+ B cells in the presence or absence of OVA_323–339 peptide and blocking antibodies to CD40L and MHC-II. Flow cytometry plots show biotin-LPETG transfer from T to B cells. Results from 2 independent experiments are summarized in the graph on the right.

Figure 2.. Bidirectional labeling of interactions between T cells and DCs in adoptive transfer models.

(A) Experimental layout for the experiments in panels (B,C). (B,C) uLIPSTIC (B) and CD40L LIPSTIC (C) labeling of adoptively-transferred DCs in an in vivo priming model. Flow cytometry plots are gated on transferred (CFSE-labeled) DCs. Results from 2 independent experiments are summarized on the graphs to the right. (D) uLIPSTIC labeling of DCs by CD8⁺ T cells. Experimental setup as in (A), but DCs were pulsed either with cognate (OVA_257–264) or control (LCMV gp33_33–41) peptides. (E-G) Labeling of antigen-specific CD4⁺ T cells by Clec9a-expressing DCs. (E) Experimental layout. (F) efficiency of recombination of the uLIPSTIC allele in migratory (m)DCs by Clec9a^Cre. (G) Left, labeling of adoptively transferred OT-II T cells upon immunization with OVA/alum. Right, summary of data from 2 independent experiments.

Figure 3.. uLIPSTIC identifies cellular partners of Treg cells, Tfh cells, and IECs.

(A) Experimental layout for panels (B,C). (B) Left, efficiency of recombination of the uLIPSTIC allele in Treg cells by Foxp3^CreERT2. Biotin signal represents the acquisition of substrate by Treg cells (the biotin-LPET-SrtA acyl intermediate) and also shows the absence of transfer of substrate to Foxp3⁻ T cells. Center, labeling of migratory (m)DCs and resident (r)DCs by Treg cells at steady state. Right, labeling of mDCs upon injection of a blocking antibody to MHC-II. (C) Summary of data from 3 independent experiments. (D) Experimental layout for panels (E,F). (E) Labeling of Tfh cells by GC B cells. Left, efficiency of recombination of the uLIPSTIC allele in GC B cells by Aicda^CreERT2 after 2 doses of tamoxifen, as in (B). Center, labeling of Tfh cells by GC B cells at 10 days after immunization with NP-OVA/alum. T cells are gated as high or low expressors of Tfh markers CXCR5 and PD-1 (Tfh^hi and Tfh^lo, respectively). Right, labeling of Tfh^hi cells upon injection of a blocking antibody to MHC-II. (F) Summary of data from 2 independent experiments. (G) Experimental layout for panels (H-K). (H) Efficiency of conversion of IECs into uLIPSTIC donors and substrate capture in Vil1-CreERT2 mice (as in (B)) and transfer to total CD45⁺ intraepithelial leukocytes. (I) Summary of data from 4 mice in 2 independent experiments. (J) Differential labeling of selected IEL populations by IEC donors. The dashed line is placed for reference. (K) Summary of data as in (I). For all column plots, each symbol represents one mouse, bars represent the mean.

Figure 4.. Using uLIPSTIC for interaction-based transcriptomics.

(A) Experimental workflow. uLIPSTIC labeling is read out using DNA-barcoded antibodies and droplet-based scRNA-seq. Data are first queried for identification of acceptor (A_n) populations, then uLIPSTIC signal within each population is correlated with expression of individual genes to search for mechanisms that drive donor-acceptor interaction. (B) UMAP plots of the CD45⁺ intraepithelial immune cell fraction from a uLIPSTIC reaction as in Fig. 3G. Data are pooled from three mice. Left, major cell populations (see Fig. S5, S6). Right, intensity of normalized uLIPSTIC signal (biotin). (C) Distribution of normalized uLIPSTIC signal among CD45⁺ cell populations. (D) UMAP plots of CD4⁺ T cells from (B). Left, major cell subpopulations (see Fig. S7). Right, intensity of normalized uLIPSTIC signal (biotin). (E) Left, inferred trajectory and right, αβTCR diversity (plotted as clone size) among CD4⁺ T cells. (F) Distribution of normalized uLIPSTIC (biotin) signal among CD4⁺ T cell subpopulations. (G) Correlation (Spearman’s ρ) between normalized uLIPSTIC signal and normalized gene expression, calculated for each gene over all CD4⁺ T cells, shown in order of increasing correlation. Selected genes are highlighted. Correlation for all selected genes was significant (FDR < 1e-23). (H) Normalized expression of selected genes. Correlation with normalized uLIPSTIC shown in parentheses. (I) Representative samples showing in vivo staining of JAML in IELs and scRNA-seq expression of Jaml in the equivalent populations. In the latter, CD8αα⁺ and γδ IEL were separated from within the “Natural IEL” cluster by the presence of rearranged αβ TCRs or expression of the Trdc gene. Fluorescent antibody was injected 6 h prior to IEL harvesting and analysis. (J) Correlation between normalized uLIPSTIC signal among all CD4⁺ T cells and expression of gene signatures up and downregulated as epithelial T cells transition from Tconv (CD4⁺CD103⁻CD8αα⁻) to CD4-IEL (CD4⁺CD103⁺CD8αα⁺) phenotypes (signatures based on data from Bilate et al.). Trend line and error are for linear regression with 95% confidence interval.