Single-cell Gene Expression With Cell Surface Protein

• Cancer Research

• Immune Research

• Disease Mechanism Research

The gel beads of 10x Feature Barcode technology have 3 capture sequences. They are: 3' expression capture sequence (poly(dT) primer sequence), surface protein library capture sequence Capture Seq 1 (Feature Barcode sequence), sequence used in CRISPR screening-Capture Seq 2. Antibody-coupled cells and gel beads form water-in-oil structures (GEMs) through the 10x Genomics platform. Next, the gel beads dissolve to release the 3 primer sequences, the cells are lysed to release the mRNA, and then the 3' expression primer and Capture Seq 1 primer capture the mRNA and protein marker sequences respectively to form cDNA, which is then used for library construction. The gene expression and surface protein expression information of the same cell can be obtained through data analysis.

10x Feature Barcode Technical Principles

10x Feature Barcode Experiment Process

A conserved dendritic-cell regulatory program limits antitumor immunity

Journal: Nature  IF: 69.504

Abstract

Checkpoint blockade therapy has improved cancer treatment, but this type of immunotherapy fails in a significant proportion of patients. Conventional type 1 dendritic cells (DC1) control response to checkpoint blockade in preclinical models and are associated with better overall survival in cancer patients, however, also in tumors resistant to checkpoint blockade DC1 was found, suggesting that the function of these cells may be altered in certain lesions. Using single-cell RNA-sequencing of human and mouse non-small cell lung cancers, the researchers identified a population of dendritic cells (DCs), termed "mature DCs rich in immunomodulatory molecules" (MREGDCs), because they coexpress immune regulatory genes (Cd274, Pdcd1lg2, and Cd200) and mature genes (Cd40, Ccr7, and Il12b). Further studies found that upregulation of IL-12 in MREGDCs was strictly dependent on interferon-γ and negatively regulated by IL-4 signaling. Blockade of IL-4 promoted IL-12 expression in tumor antigen-bearing mregDC1s, expanding the tumor-infiltrating effector T cell repertoire and reducing tumor burden.

Main Conclusions

1. Sorting, single-cell RNA-sequencing and CITE-seq of DCs from normal and tumor-bearing mouse lung tissues revealed a special subset of DCs associated with non-small cell lung cancer, named mregDCs; the same subpopulation type was also found in other data.

DC cell grouping

Subgroup characteristic gene expression

The expression of cell surface proteins in each subgroup

Figure 1. Single-cell transcriptome sequencing and surface protein detection identify three DC subsets

2. Using fluorescence imaging and transcriptome sequencing, it was found that the initiation of the mregDC program in DC1 was associated with the uptake of tumor antigens; moreover, blocking IL-4 signaling can enhance DC1 function and antitumor immunity.

DC1 co-localizes with tumor antigens

Blocking IL-4 reduces tumor size and induces T cell expansion

Figure 2. Research on the function and signaling pathway of MregDC

3. mregDCs were also found in human samples, and the cells were concentrated in the injured area.

Cell grouping

The expression of each subgroup gene set

Comparison of mregDC characteristic gene expression in human and mouse

Figure 3. Single-cell sequencing validation in human samples