Background

Immunotherapy has proven clinical efficacy and has tremendous potential, but actual clinical benefit is experienced by only a small subset of patients. There is a growing necessity of additional research to improve beneficial clinical outcomes. It is particularly important to analyze steady-state anti-tumor immunity and monitor the effects of therapy on the tumor microenvironment, including tumor-infiltrating leukocytes (TILs). However, TIL numbers can be very low and small subpopulations might escape analysis when they are lost in background noise. This can be especially challenging when performing single-cell analysis. Moreover, a prerequisite for single-cell analysis is a debris free single-cell suspension with a viability above 80%.

Here we demonstrate, that our workflow solution, which includes tissue storage, tumor dissociation, dead cell removal, and cell isolation, yields enriched viable T and B cell populations that significantly increase single-cell immune profiling results.

Results

Immunophenotyping of TILs by flow cytometry reveals low T and B cell frequencies

In the dissociated bulk tumor sample only 14% of the cells were leukocytes. Using the appropriate gating strategy, leukocytes were further subclassified as T cells, B cells, neutrophils, eosinophils, and NK cells.

Magnetic isolation of TILs increases the sensitivity of single-cell receptor sequencing and immunoprofiling

For the single-cell immune profiling assay, the frozen tumor cell suspension was thawed and dead cells were removed. The percentage of viable cells in the sample was increased from only 26% prior to dead cell removal to over 80%. After dead cell removal, T cells were enriched to over 80% using REAlease® CD4/CD8 (TIL) MicroBeads (fig. 3). 

The clonality of TILs was assessed via single-cell receptor sequencing, either performed with unseparated bulk cells or with isolated TILs. The comparison proved that the magnetic isolation of TILs improved the resolution of the RNA sequencing, highlighting clonotypes otherwise poorly or altogether unrepresented. In figure 4, the top 50 TCRβ (A) and BCR (B) CDR3 clonotypes identified via RNA sequencing in the isolated TIL population (purple) are represented together with the corresponding number of cells. The red bars show the number of cells that showed the same TCRβ (A) and BCR (B) CDR3 clonotypes in the bulk sample.