The identification and functional analysis of CD8+PD-1+CD161+ T cells in hepatocellular carcinoma
Immunotherapy is a powerful therapeutic strategy for end-stage hepatocellular carcinoma (HCC). It is well known that T cells, including CD8+PD-1+ T cells play important roles involving tumor development. However, their underlying phenotypic and functional differences of T cell subsets remain unclear. We constructed single-cell immune contexture involving approximate 20,000,000 immune cells from 15 pairs of HCC tumor and non-tumor adjacent tissues and 10 blood samples (including 5 of HCCs and 5 of healthy controls) by mass cytometry. scRNA-seq and functional analysis were applied to explore the function of cells. Multi-color fluorescence staining and tissue microarrays were used to identify the pathological distribution of CD8+PD-1+CD161+/- T cells and their potential clinical implication. The differential distribution of CD8+ T cells subgroups was identified in tumor and non-tumor adjacent tissues. The proportion of CD8+PD1+CD161+ T cells was significantly decreased in tumor tissues, whereas the ratio of CD8+PD1+CD161- T cells was much lower in non-tumor adjacent tissues. Diffusion analysis revealed the distinct evolutionary trajectory of CD8+PD1+CD161+ and CD8+PD1+CD161- T cells. scRNA-seq and functional study further revealed the stronger immune activity of CD8+PD1+CD161+ T cells independent of MHC class II molecules expression. Interestingly, similar change of the ratio of CD8+CD161+/ CD8+CD161- T cells was also found in peripheral blood samples collected from HCC cases, indicating their potential usage clinically. We here identified different distribution, function and trajectory of CD8+PD-1+CD161+ and CD8+PD-1+CD161- T cells in tumor lesions, which provided new insights for the heterogeneity of immune environment in HCCs and also shed light on the potential target for immunotherapy.
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Fifteen groups of TILs and NILs and ten samples of leukocytes from peripheral blood were obtained as described above and subjected to CyTOF analysis. A total of 35 immune antibodies were used to distinguish immune cell subsets. Preconjugated antibodies are purchased directly from Biolegend, USA. Leukocytes were washed and stained with 10 mM cisplatin for 2 minutes to identify live/dead cells, and incubated with metal-conjugated surface-membrane antibodies for 30 minutes at 37 ºC. The cells were then fixed and added to a cell intercalation (mixture of fix perm buffer and iridium) for overnight. Finally, visual analysis was performed with Helios mass cytometer (Fludigm, USA). We used CountessII Automated Cell Counter (Thermo Fisher Scientific, USA) to count cells waiting to be tested and adjusted concentration to an ideal concentration of 1 × 10^6/ ml. Then, cDNA was marked by 10x GemCode Technology. Gel beads containing barcode information were first mixed with cells and enzymes. Droplets flowed into reservoir and were collected and then dissolved and released primer sequences for reverse transcription. cDNA was used as templates to amplify PCR. A sequencing library was constructed by mixing products containing barcode amplification information in each droplet. First, DNA fragments were broken into 200-300 BP fragments by Biorupter Ultrasound Fragmentation Instrument. Next, DNA library was amplified by PCR with sequencing connector P5 and sequencing primer R1. Finally, prepared samples were subjected to the 10Х single-cell sequencing analysis platform.