title: Improvement of IFNg ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis.
abstract: Immune monitoring of functional responses is a fundamental parameter to establish correlates of protection in clinical trials evaluating vaccines and therapies to boost antigen-specific responses. The IFNg ELISPOT assay is a well-standardized and validated method for the determination of functional IFNg-producing T-cells in peripheral blood mononuclear cells (PBMC); however, its performance greatly depends on the quality and integrity of the cryopreserved PBMC. Here, we investigate the effect of overnight (ON) resting of the PBMC on the detection of CD8-restricted peptide-specific responses by IFNg ELISPOT. The study used PBMC from healthy donors to evaluate the CD8 T-cell response to five pooled or individual HLA-A2 viral peptides. The results were analyzed using a modification of the existing distribution free resampling (DFR) recommended for the analysis of ELISPOT data to ensure the most rigorous possible standard of significance. The results of the study demonstrate that ON resting of PBMC samples prior to IFNg ELISPOT increases both the magnitude and the statistical significance of the responses. In addition, a comparison of the results with a 13-day preculture of PBMC with the peptides before testing demonstrates that ON resting is sufficient for the efficient evaluation of immune functioning.
authors.affiliation: Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway Port St. Lucie, FL 34987, USA. rsantos@tpims.org.
authors.forename: Radleigh
authors.initials: R
authors.lastname: Santos
authors.affiliation: Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121, USA. abunying@tpims.org.
authors.forename: Alcinette
authors.initials: A
authors.lastname: Buying
authors.affiliation: Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121, USA. nsabri@tpims.org.
authors.forename: Nazila
authors.initials: N
authors.lastname: Sabri
authors.affiliation: Cedars-Sinai, 8700 Beverly Blvd., Los Angeles, CA 90048, USA. John.Yu@cshs.org.
authors.forename: John
authors.initials: J
authors.lastname: Yu
authors.affiliation: ImmunoCellular Therapeutics Ltd., 23622 Calabasas Road, Calabasas, CA 91302, USA. anthony.gringeri@imuc.com.
authors.forename: Anthony
authors.initials: A
authors.lastname: Gringeri
authors.affiliation: ImmunoCellular Therapeutics Ltd., 23622 Calabasas Road, Calabasas, CA 91302, USA. james.bender@lionbio.com.
authors.forename: James
authors.initials: J
authors.lastname: Bender
authors.affiliation: ZellNet Consulting, 555 North Ave., Fort Lee, NJ 07024, USA. sylvia@zellnet.com.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Janetzki
authors.affiliation: Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121, USA. pinilla@tpims.org.
authors.forename: Clemencia
authors.initials: C
authors.lastname: Pinilla
authors.affiliation: Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121, USA. vjudkowski@tpims.org.
authors.forename: Valeria A
authors.initials: VA
authors.lastname: Judkowski