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

Abstract

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.

QuantiFERON-CMV as a Predictor of CMV Events During Preemptive Therapy in CMV-seropositive Kidney Transplant Recipients

“…54 On the other side, because ELISpot testing showed higher IFN-γ production with interleukin-2 costimulation in an experimental study, basiliximab might presumably interfere with QFCMV results. 27 This study followed most Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis 55 recommendations. Nevertheless, our study has some weaknesses.…”

“…Variables such as recipient/donor age and sex, type of donor, delayed graft function, and acute rejection were selected on the basis of previous publications 2 and availability of data in the routine care of these patients. The following potential predictors were obtained around day 30 (accepted range, days [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40]: immunosuppressive medication dosing, lymphocyte counts, 12 complement C3, total IgG levels, 13 and estimated GFR (eGFR). 14 Recipient HLA-A2, number of HLA mismatches (loci A, B, and DR only), extended criteria donor, kidney donor risk index, and cold ischemia time were also tested as predictors.…”

Current Trends in Neoantigen-Based Cancer Vaccines

“…If the pMHC is recognized by the TCR, the cytokines released by the T cells are captured by the antibodies and the fluorescence created by the enzyme-substrate reaction can be detected. The extent of T-cell activation is justified by the depth of the fluorescence at the bottom of the plate [ 45 , 46 ]. A shortcoming of this method is that reagents are expensive for high-throughput screening.…”

A Multiple-Target Simultaneous Detection Method for Immunosorbent Assay and Immunospot Assay

“…Immunolabeling technology refers to the use of fluorescence, radioisotopes, enzymes, luminescent agents, or electron-dense substances as tracers to label antibodies or antigens for detecting or imaging. Enzyme-linked immunosorbent assay (ELISA) is one of the most widely used methods to quantitatively measure antigens or antibodies in fluid samples, such as in serum. − Enzyme-linked immunospot (ELISPOT) is a type of assay that focuses on quantitatively measuring the frequency of cytokine secretion from every single cell in cultured media. − The ELISPOT assay is also a form of immunostaining since it is classified as a technique that uses antibodies to detect a protein analyte. Because of the single-cell level detection, ELISPOT is sensitive and can detect one cell to several cells secreting detected protein from approximately 4 × 10 4 to 6 × 10 5 cells. , However, the traditional ELISA and ELISPOT technology have some limitations.…”

“…One limitation is that these traditional methods are not sensitive enough to visualize low abundance targets. ,,,− If we could improve the sensitivity of traditional ELISA and ELISPOT, then we would broaden the applications of these methods. Incorporating a signal amplification system that can further improve the sensitivity of traditional ELISA and ELISPOT is hence critically needed to generate a higher signal for target detection.…”