The co-stimulation of anti-CD28 and IL-2 enhances the sensitivity of ELISPOT assays for detection of neoantigen-specific T cells in PBMC

Neoantigen-based cancer immunotherapies hold the promise of being a truly personalized, effective treatment for diverse cancer types. ELISPOT assays, as a powerful experimental technique, can verify the existence of antigen specific T cells to support basic clinical research and monitor clinical trials. However, despite the high sensitivity of ELISPOT assays, detecting immune responses of neoantigen specific T cells in a patient or healthy donor's PBMCs is still extremely difficult, since the frequency of these T cells can be very low. We developed a novel experimental method, by co-stimulation of T cells with anti-CD28 and IL-2 at the beginning of ELISPOT, to further increase the sensitivity of ELISPOT and mitigate the challenge introduced by low frequency T cells. Under the optimal concentration of 1 μg/ml for anti-CD28 and 1 U/ml for IL-2, an 11.7-fold increase of T cell response against CMV peptide was observed by using our method, and it outperforms other cytokine stimulation alternatives (5–10 folds). We also showed that this method can be effectively applied to detect neoantigen-specific T cells in healthy donors' and a melanoma patient's PBMCs. To the best of our knowledge, this is the first report that the co-stimulation of anti-CD28 and IL-2 is able to significantly improve the sensitivity of ELISPOT assays, indicating that anti-CD28 and IL-2 signaling can act in synergy to lower the T cell activation threshold and trigger more neoantigen-specific T cells.