Strategies for optimizing CITE-seq for human islets and other tissues

Abstract

Defining the immunological landscape of human tissue is an important area of research, but challenges include the impact of tissue disaggregation on cell phenotypes and the low abundance of immune cells in many tissues. Here, we describe methods to troubleshoot and standardize Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) for studies involving enzymatic digestion of human tissue. We tested epitope susceptibility of 92 antibodies commonly used to differentiate immune lineages and cell states on human peripheral blood mononuclear cells following treatment with an enzymatic digestion cocktail used to isolate islets. We observed CD4, CD8a, CD25, CD27, CD120b, CCR4, CCR6, and PD1 display significant sensitivity to enzymatic treatment, effects that often could not be overcome with alternate antibodies. Comparison of flow cytometry-based CITE-seq antibody titrations and sequencing data supports that for the majority of antibodies, flow cytometry accurately predicts optimal antibody concentrations for CITE-seq. Comparison by CITE-seq of immune cells in enzymatically digested islet tissue and donor-matched spleen not treated with enzymes revealed little digestion-induced epitope cleavage, suggesting increased sensitivity of CITE-seq and/or that the islet structure may protect resident immune cells from enzymes. Within islets, CITE-seq identified immune cells difficult to identify by transcriptional signatures alone, such as distinct tissue-resident T cell subsets, mast cells, and innate lymphoid cells (ILCs). Collectively this study identifies strategies for the rational design and testing of CITE-seq antibodies for single-cell studies of immune cells within islets and other tissues.

Keywords: CITE-seq; flow cytometry; pancreas; single cell RNA seq; tissue immunity.

Figure 1

Flow cytometry reveals T cell specific, deleterious effects of islet digestive enzymes on staining of antibody clones used for generation of TotalSeq-C oligo-antibodies. (A) Activated or non-activated PBMCs were treated with a digestive enzyme solution and stained with flow cytometry antibodies corresponding to clones used in the TotalSeq-C commercial antibody catalogue of oligo-antibodies used for CITE-seq. (B) Markers were selected to identify key cellular subsets of T lymphocytes, monocytes, and innate lymphoid cells, in addition to other antibodies used for e.g., leukocyte selection, stress indicators, and immune activation.

Figure 2

UpSet plot of clones included in the study, organized by immune cell lineage (T cell, NK/ILC, or monocyte).(A) Relative proportions of sensitive, partially sensitive, and insensitive clones per grouping. (B) Histogram representing number of clones tested per grouping. (C, left) From top to bottom, total number of clones tested for markers that are expressed by T cells, NK/ILCs, and monocytes, respectively. Line plot (right) signifies groupings of markers for data in (A, B) that are expressed by one, two, or all three cell types, as indicated by connected dots (e.g., the first column describes data for markers that are expressed by all three cell types, the second column for markers expressed only by T cells and NK/ILCs, etc.). Groupings are exclusive.

Figure 3

The effects of enzymatic digestion on key phenotypic markers of concern in the TotalSeq-C antibody library. White and blue circles indicate pre- and post-digestion values, respectively. Each pairing signifies an independent experimental replicate of pre- and post-treatment measurements, and the proportion of cells positive for the marker of interest was determined using standard flow gating. Parent cells were total live lymphocytes after applying quality control gating as outlined in Supplemental Figure S1 .

Figure 4

Circle packing heat map of clone sensitivity to digestive enzymes. The colour scale indicates relative proportion of replicates sensitive or partially sensitive to digestion, and the size of each circle indicates the number of replicates included in analysis. Circle containers indicate successive levels of hierarchy, where clones are grouped according to immune marker (the colour of these encapsulating hierarchy circles, which contain multiple daughter clones, is not indicative of daughter clone sensitivity).

Figure 5

Titrations of flow cytometry antibodies corresponding to oligo-antibody clones in the TotalSeq-C catalogue reveals optimal staining conditions. PBMCs were stained using PE antibodies against the marker of interest and performed in 3-fold serial dilutions. Dilution chosen is shown in blue. (A) Example titrations showing percent positive noise + signal and percent positive signals. The titration chosen is the dilution that does not lose positive signal yet has the least signal + noise percent positive. (B) 3-point titrations performed on markers affected by enzymatic digestion. (C) Example legend for histograms shown in B, where the top plot is titration #1 and the lowest dilution, middle plot is titration #2 and the middle concentration, and the bottom plot is titration #3 and highest concentration of antibody.

Figure 6

Workflow and combined islet and spleen lineage maps. (A) Example workflow of CITE-seq. Spleen and islet samples were enriched for CD45⁺ immune cells. Immune cells from spleen were stained with oligo-antibody conjugated TotalSeqC antibodies and sent for 10x sequencing. Islet immune cells were either were stained with oligo-antibody conjugated TotalSeqC antibodies or stained for both oligo-antibody conjugated TotalSeqC and flow cytometry antibodies at the same time and FACS sorted to enrich for NK cells and ILCs and then sent for 10x sequencing. (B) UMAPs of islet cell types, islet cell origin (FACS sorted or CD45-enriched), and spleen cell types. (C) Merged islet and spleen lineage, cell types and tissue of origin UMAPs. (D) Composition of cell types from each organ. (E) T, NK and ILC lineage markers. (F) Myeloid lineage markers. (G) B cell, plasma cell and mast cell lineage markers.

Figure 7

CITE-Seq protein expression is validated by flow cytometry on healthy human islets. Healthy human islets were analyzed by CITE-seq and flow cytometry. Surface marker proteins found to be expressed by islet-resident immune cells by CITE-seq were analyzed by flow cytometry in order to validate CITE-seq findings. PBMCs were used a staining control for flow cytometry analysis. (A) human islet UMAP, (B) Myeloid population analysis of CD14, CD206 and HLA-DR expression. (C) T cell population analysis of CD4, CD8, CD45Ro, CCR4 and CD103. (D) ILC analysis of CD56 and CD127.

Figure 8

CITE-seq allows identification cell types through the expression of surface markers not captured by single cell RNA sequencing. (A) RNA (genes shown in green) and protein (surface markers shown in blue) expression of matched molecules. (B) Markers shown to be sensitive to digestion by flow cytometry on T cells. T cells subsets were extracted from the combined islet/spleen objects and (C) titration markers were compared between the digested (islets) and non-digested (spleen) samples.