Challenges of the inflammatory microenvironment

Antigen-presenting cells (APCs) and T cells are present in many solid tumor types, where they are critical determinants of tumor development and disease outcome. A hallmark of immune-infiltrate human tumor tissues is the presence of an inflammatory microenvironment; however, it is not well understood which immune cell subsets and signaling pathways are unique to the human tumor microenvironment compared to those from general inflammatory processes.

Here we summarize the results from a recent publication from the Mair lab, which hypothesized that comparing the human tumor microenvironment with non-malignant, inflamed tissues could identify tumor-unique immune alterations.

Check out our previous interview with the author, Florian Mair.

Methods

To identify tumor-enriched immune alterations, several single-cell analysis pipelines were combined to generate a comprehensive immune landscape of human head and neck squamous cell carcinomas (HNSCC). This immune landscape was then compared with site-matched non-malignant inflamed oral mucosal (OM) tissue. Matched peripheral blood samples were collected and cryopreserved peripheral blood mononuclear cells (PBMCs) from healthy controls were used as a longitudinal technical control for flow cytometry acquisitions.

To phenotype the congruence of HNSCC and OM tissue, two flow cytometry panels comprised of 30 parameters and computational analysis were used to identify specific immune subset differences. To better understand the immune alterations observed, the author used NicheNet to predict enriched ligand-receptor interactions between APCs and Treg cells in HNSCC tissue. To understand the functional capacity of specific immune cells, classic stimulation and suppression assays and bulk-RNA sequencing were performed.

Flow cytometry was used to confirm uniqueness of immune subsets to HNSCC tissue compared to OM. Flow cytometric analysis followed best practices outlined for use of flow cytometry and suggestions for data analysis. Publicly available scRNA-seq datasets were mined to assess uniqueness compared to other tumor types.

Summary of key findings

The analysis revealed a large overlap in the composition and phenotype of immune cells in HNSCC and inflamed OM tissue.

Computational analysis approaches identified tumor-specific changes in subsets of activated APCs and Treg cells. It predicted that Treg cells in the tumor actively received T cell receptor (TCR) signals and responded to proinflammatory cytokines IL-1 and IL-18. IL-1 receptor 1 (IL1R1) was expressed by tumor-infiltrating Treg cells but not by tumor-infiltrating CD4+ T cells, CD8+ T cells or T cells in the peripheral blood.

The functional capacity of IL1R1+ Treg cells was assessed through comparison with their IL1R1− counterparts using a transcriptomics approach to measure the expression of genes with known relevance to the immune system. This demonstrated that intertumoral IL1R1+ Treg cells showed substantial clonal expansion, superior immunosuppressive function and hallmarks of recent TCR stimulation.

This Treg population was uniquely identifiable through the co-expression of IL1R1 and inducible T cell co-stimulator (ICOS), with IL1R1+ICOS+ Treg cells significantly enriched in HNSCC compared with OM. However, expression of IL1R1 transcript was detected across 19 different tumor types so was not unique to HNSCC.

Conclusion

Findings of the present paper show that the co-expression of IL1R1 and ICOS uniquely identifies an intertumoral Treg population from all other hematopoietic derived cells in the tumor or peripheral blood. The outlined approach, using flow cytometry, could serve as a blueprint to identify immunological congruencies and improve our understanding of disease-specific processes. 

Read the full article: Extricating human tumor immune alterations from tissue inflammation - PMC (nih.gov)