A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools

Personalized vaccines against patient-unique tumor-associated antigens represent a promising new approach for cancer immunotherapy. Vaccine efficacy is assessed by quantification of changes in the frequency and/or the activity of antigen-specific T cells. Enzyme-linked immunosorbent spot (ELISpot) and flow cytometry (FCM) are methodologies frequently used for assessing vaccine efficacy. We tested these methodologies and found that both ELISpot and standard FCM [monitoring CD3/CD4/CD8/IFN gamma/Viability+CD14+CD19 (dump)] demonstrate background IFN gamma secretion, which, in many cases, was higher than the antigen-specific signal measured by the respective methodology (frequently ranging around 0.05-0.2%). To detect such weak T-cell responses, we developed an FCM panel that included two early activation markers, 4-1BB (CD137) and CD40L (CD154), in addition to the above-cited markers. These two activation markers have a close to zero background expression and are rapidly upregulated following antigen-specific activation. They enabled the quantification of rare T cells responding to antigens within the assay well. Background IFN gamma-positive CD4 T cell frequencies decreased to 0.019% +/- 0.028% and CD8 T cells to 0.009% +/- 0.013%, which are 19 and 13 times lower, respectively, than without the use of these markers. The presented methodology enables highly sensitive monitoring of T-cell responses to tumor-associated antigens in the very low, but clinically relevant, frequencies.

Automated summary

Personalized vaccines for cancer immunotherapy can be evaluated by quantifying changes in antigen-specific T cell frequency or activity using ELISpot and FCM. However, background IFN gamma secretion can interfere with the accuracy of these methods. To overcome this, we developed an FCM panel including early activation markers to enable highly sensitive monitoring of low-frequency T cell responses to tumor-associated antigens.