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

By Yunxia Tang, Linnan Zhu, Qumiao Xu, Xiuqing Zhang, Bo Li, Leo J Lee

Posted 08 Sep 2019
bioRxiv DOI: 10.1101/760355 (published DOI: 10.1016/j.jim.2020.112831)

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 1μg/mL for anti-CD28 and 1U/ml for IL-2, our new method can increase sensitivity by up to 5 folds comparing to a conventional ELISPOT, and outperforms other cytokine stimulation alternatives. 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.

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