Performance and Stability of New Class of Fetal Bovine Sera (FBS) and Its Lyophilized Form in ELISpot and FluoroSpot Assays: Applications for Monitoring the Immune Response in Vaccine, and Cell and Gene Immunotherapy in Clinical Trials

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2024 Mar 19

PMID 38502401

Authors

Zhinous Hosseini

Christopher J Groves

Penny Anders

Kristen Cave

Madelyn Krunkosky

Brandi Chappell

Sofie Pattyn

Devin Davis

Sylvia Janetzki

Elizabeth Reap

Affiliations

Soon will be listed here.

Abstract

Interferon-gamma (IFNγ) ELISpot and FluoroSpot are widely used assays to detect functional cell responses in immunotherapy clinical studies. Recognized for their importance in vaccine development studies to quantitate immune responses, these assays have more recently risen to the forefront in cell and gene therapy as well as cancer immunotherapy fields where responses against cancer neoantigens are not easily detectable above assay background. Here, we test a new class of fetal bovine serum (FBS), CultraPure FBS, in ex vivo ELISpot and FluoroSpot assays and cultured FluoroSpot assays following in vitro expansion. Several CultraPure FBS lots that have been specially formulated through the process of lyophilization (lyo-FBS) were compared to liquid CultraPure FBS. We stimulated human PBMCs with antigen-specific peptide pools diluted in media supplemented with liquid CultraPure FBS or lyo-FBS and found equivalent cytokine production with negligible to no assay background with both liquid and lyo-FBS formats. Moreover, the lyo-FBS showed lot-to-lot consistency and 90-day refrigerated (4 °C) stability in both ex vivo direct and in vitro cultured assays. In addition, we present here a method using lyo-FBS for the expansion of low-frequency antigen-specific T cells, mimicking the low frequency seen with cancer neoantigens by utilizing a cultured FluoroSpot assay. Our results demonstrate the presence of Granzyme B, interferon-gamma (IFNγ), and tumor necrosis factor (TNF) production by antigen-specific polyfunctional T cells following a 9-day culture using media supplemented with lyo-FBS.

Citing Articles

Polyfunctional T cells and unique cytokine clusters imprint the anti rAAV2/rAAV9 vector immune response.

Holtkamp S, Lagoda F, Lister A, Harish P, Kleymann U, Pesch T Front Immunol. 2024; 15:1450524.

PMID: 39654900 PMC: 11625739. DOI: 10.3389/fimmu.2024.1450524.

References

Czerkinsky C, Nilsson L, Nygren H, OUCHTERLONY O, Tarkowski A . A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunol Methods. 1983; 65(1-2):109-21. DOI: 10.1016/0022-1759(83)90308-3. View

Reap E, Dryga S, Morris J, Rivers B, Norberg P, Olmsted R . Cellular and humoral immune responses to alphavirus replicon vaccines expressing cytomegalovirus pp65, IE1, and gB proteins. Clin Vaccine Immunol. 2007; 14(6):748-55. PMC: 1951075. DOI: 10.1128/CVI.00037-07. View

Patton K, Harrison M, Long B, Lau K, Holcomb J, Owen R . Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method. Mol Ther Methods Clin Dev. 2021; 22:183-195. PMC: 8399379. DOI: 10.1016/j.omtm.2021.05.012. View

Arnaud M, Chiffelle J, Genolet R, Rodrigo B, Perez M, Huber F . Sensitive identification of neoantigens and cognate TCRs in human solid tumors. Nat Biotechnol. 2021; 40(5):656-660. PMC: 9110298. DOI: 10.1038/s41587-021-01072-6. View

Kalyuzhny A, Stark S . A simple method to reduce the background and improve well-to-well reproducibility of staining in ELISPOT assays. J Immunol Methods. 2001; 257(1-2):93-7. DOI: 10.1016/s0022-1759(01)00451-3. View

Janetzki S, Price L, Britten C, van der Burg S, Caterini J, Currier J . Performance of serum-supplemented and serum-free media in IFNgamma Elispot Assays for human T cells. Cancer Immunol Immunother. 2009; 59(4):609-18. PMC: 2813531. DOI: 10.1007/s00262-009-0788-2. View

Janetzki S, Price L, Schroeder H, Britten C, Welters M, Hoos A . Guidelines for the automated evaluation of Elispot assays. Nat Protoc. 2015; 10(7):1098-115. DOI: 10.1038/nprot.2015.068. View

Janetzki S, Panageas K, Ben-Porat L, Boyer J, Britten C, Clay T . Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI). Cancer Immunol Immunother. 2007; 57(3):303-15. PMC: 2150634. DOI: 10.1007/s00262-007-0380-6. View

van der Valk J, Bieback K, Buta C, Cochrane B, Dirks W, Fu J . Fetal Bovine Serum (FBS): Past - Present - Future. ALTEX. 2017; 35(1):99-118. DOI: 10.14573/altex.1705101. View

10.

Romer P, Berr S, Avota E, Na S, Battaglia M, Ten Berge I . Preculture of PBMCs at high cell density increases sensitivity of T-cell responses, revealing cytokine release by CD28 superagonist TGN1412. Blood. 2011; 118(26):6772-82. DOI: 10.1182/blood-2010-12-319780. View

11.

Wegner J, Hackenberg S, Scholz C, Chuvpilo S, Tyrsin D, Matskevich A . High-density preculture of PBMCs restores defective sensitivity of circulating CD8 T cells to virus- and tumor-derived antigens. Blood. 2015; 126(2):185-94. DOI: 10.1182/blood-2015-01-622704. View

12.

Kutscher S, Dembek C, Deckert S, Russo C, Korber N, Bogner J . Overnight resting of PBMC changes functional signatures of antigen specific T- cell responses: impact for immune monitoring within clinical trials. PLoS One. 2013; 8(10):e76215. PMC: 3795753. DOI: 10.1371/journal.pone.0076215. View

13.

Santos R, Buying A, Sabri N, Yu J, Gringeri A, Bender J . Improvement of IFNg ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis. Cells. 2014; 4(1):1-18. PMC: 4381205. DOI: 10.3390/cells4010001. View

14.

Moodie Z, Price L, Gouttefangeas C, Mander A, Janetzki S, Lower M . Response definition criteria for ELISPOT assays revisited. Cancer Immunol Immunother. 2010; 59(10):1489-501. PMC: 2909425. DOI: 10.1007/s00262-010-0875-4. View

15.

Moodie Z, Price L, Janetzki S, Britten C . Response determination criteria for ELISPOT: toward a standard that can be applied across laboratories. Methods Mol Biol. 2011; 792:185-96. DOI: 10.1007/978-1-61779-325-7_15. View