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

SIMPLE SUMMARY: Personalized anti-cancer vaccines utilize peptides derived from mutated proteins (neoantigens) or from proteins aberrantly expressed by the cancer cells (e.g., cancer testis antigens). Vaccination increases the frequency of anti-tumoral T cells and promotes their antigen-specific activation. Current methods used for identifying peptides which have elicited T-cell responses, do not measure these responses with sufficient sensitivity. Non-antigen-specific background activation frequency of currently applied methodologies ranges from 0.1–0.7%, while the frequencies of antigen-specific responses to vaccinated peptides are often at the lower end of this range (0.05–0.2%). As a result, many peptides which have succeeded in eliciting a response are not detected. We developed a methodology that can identify response frequencies as low as 0.01–0.02%, thereby facilitating the detection of T-cell responses that would otherwise be missed. Our method enables more accurate monitoring of the dynamic changes in vaccination-induced T-cell responses and thereby more efficacious design of personalized vaccines. ABSTRACT: 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/IFNγ/Viability+CD14+CD19 (dump)] demonstrate background IFNγ 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 IFNγ-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.