AccScience Publishing / JBM / Online First / DOI: 10.14440/jbm.0122
Submit to JBM
Cite this article
13
Download
287
Views
Journal Browser
Volume | Year
Issue
Search
Forthcoming Issue
Current Issue
View All
News and Announcements
View All
RESEARCH ARTICLE

Modelling innate and adaptive immune responses in whole blood: A modified ex vivo assay without anticoagulants and synthetic media

Victor I. Seledtsov1* ,  Tatyana Y. Lyubavskaya1 ,  Anatoly A. Pyshenko1 ,  Alexei von Delwig1 ,  Irina A. Seledtsova1
Show Less
1 Russian Scientific Centre for Surgery named after Academician B. V. Petrovsky, Federal State Budgetary Scientific Institution, Moscow 119991, Russia
JBM, e99010076 https://doi.org/10.14440/jbm.0122
Submitted: 6 May 2025 | Revised: 13 August 2025 | Accepted: 19 August 2025 | Published: 10 September 2025
© 2025 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
Download PDF
Cite
XML
Abstract

Background: Blood is central to immune defense, rendering accurate assessment of its immunoreactivity vital for medical and biotechnological applications. Objective: This study presented a novel whole blood immunoreactivity assay (WBIA) designed to mimic natural physiological conditions, preserving essential cell–cell and cell–cytokine interactions for ex vivo immunological analysis. Methods: Fresh whole blood (with or without heparin) was stimulated with lipopolysaccharide (LPS), concanavalin A (Con A), or both, activating innate and adaptive immunity. Cytokine levels were measured through enzyme-linked immunosorbent assay after incubation. Results: Coagulation enhanced secretion of interleukin (IL)-2 and vascular endothelial growth factor (VEGF) in mitogen-stimulated samples. LPS induced tumor necrosis factor (TNF)-α, IL-6, and VEGF, while LPS + Con A co-stimulation produced the highest levels of interferon (IFN)-γ, IL-2, and IL-10. Peak cytokine concentrations were reached at 18 h, declining by 48–72 h. In 18 h LPS + Con A-stimulated serum blood samples from 30 healthy donors (19 women, 11 men, aged 30–55), cytokine levels (pg/mL, mean ± standard error of the mean) were as follows: IL-1β at (521 ± 62), IL-2 (24 ± 4), IL-6 (569 ± 43), IL-8 (277 ± 28), IL-10 (198 ± 35), IL-18 (293 ± 19), IFN-γ (227 ± 108), TNF-α (930 ± 126), and VEGF (655 ± 55). Conclusion: The WBIA provides a reliable, physiologically relevant model for evaluating immune responses to stimuli. Its high fidelity to in vivo conditions makes it a valuable tool for testing immunomodulatory drugs and monitoring immune status in clinical settings.

Keywords
Whole blood immunoreactivity assay
Innate immunity
Adaptive immunity
Cytokines
Immune status
Funding
This research was supported by the Ministry of Science and Higher Education of the Russian Federation (“Extracorporeal auto-immunotherapy of chronic diseases of the musculoskeletal system”, grant number FURG-2023- 0074 and “Application of blood components with specified physicochemical and immunobiological properties in the treatment of wound and traumatic injuries of the body”, grant number FUGR-2023-77).
Conflict of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
References
  1. Müller S, Kröger C, Schultze JL, Aschenbrenner AC. Whole blood stimulation as a tool for studying the human immune system. Eur J Immunol. 2024;54(2):e2350519. doi: 10.1002/eji.202350519

 

  1. Härtel C, Bein G, Kirchner H, Klüter H. A human whole-blood assay for analysis of T-cell function by quantification of cytokine mRNA. Scand J Immunol. 1999;49(6):649-654. doi: 10.1046/j.1365-3083.1999.00549.x

 

  1. Augustine NH, Pasi BM, Hill HR. Comparison of ATP production in whole blood and lymphocyte proliferation in response to phytohemagglutinin. J Clin Lab Anal. 2007;21(5):265-270. doi: 10.1002/jcla.20182

 

  1. Feuerecker M, Mayer W, Kaufmann I, et al. A corticoid-sensitive cytokine release assay for monitoring stress-mediated immune modulation. Clin Exp Immunol. 2013;172(2):290-299. doi: 10.1111/cei.12049

 

  1. Tran TAT, Grievink HW, Lipinska K, et al. Whole blood assay as a model for in vitro evaluation of inflammasome activation and subsequent caspase-mediated interleukin-1 beta release. PLoS One. 2019;14(4):e0214999. doi: 10.1371/journal.pone.0214999

 

  1. Buchheim JI, Feuerecker M, Balsamo M, et al. Monitoring functional immune responses with a cytokine release assay: ISS flight hardware design and experimental protocol for whole blood cultures executed under microgravity conditions. Front Physiol. 2024;14:1322852. doi: 10.3389/fphys.2023.1322852

 

  1. Gervais A, Le Floc’h C, Le Voyer T, et al. A sensitive assay for measuring whole-blood responses to type I IFNs. Proc Natl Acad Sci U S A. 2024;121(40):e2402983121. doi: 10.1073/pnas.2402983121

 

  1. Muñoz EM, Linhardt RJ. Heparin-binding domains in vascular biology. Arterioscler Thromb Vasc Biol. 2004;24(9):1549-1557. doi: 10.1161/01.atv.0000137189.22999.3f

 

  1. Xu X, Dai Y. Heparin: An intervenor in cell communication. J Cell Mol Med. 2010;14(1-2):175-180. doi: 10.1111/j.1582-4934.2009.00871.x

 

  1. Łukasik ZM, Makowski M, Makowska JS. From blood coagulation to innate and adaptive immunity: The role of platelets in the physiology and pathology of autoimmune disorders. Rheumatol Int. 2018;38(6):959-974. doi: 10.1007/s00296-018-4001-9

 

  1. Costantini TW, Kornblith LZ, Pritts T, Coimbra R. The intersection of coagulation activation and inflammation after injury: What you need to know. J Trauma Acute Care Surg. 2024;96(3):347-356. doi: 10.1097/TA.0000000000004190

 

  1. Seledtsov VI, Pyshenko AA, Lyubavskaya TY, Seledtsova IA, Von Delwig AA. Blood coagulation favors anti-Inflammatory immune responses in whole blood. Hematol Rep. 2025;17(2):19. doi: 10.3390/hematolrep17020019

 

  1. Petrovsky N, Harrison LC. Cytokine-based human whole blood assay for the detection of antigen-reactive T cells. J Immunol Methods. 1995;186(1):37-46. doi: 10.1016/0022-1759(95)00127-v

 

  1. Otani N, Shima M, Ueda T, et al. Evaluation of influenza vaccine-induced cell-mediated immunity: Comparison between methods using peripheral blood mononuclear cells and whole blood. Microbiol Immunol. 2019;63(6):223-228. doi: 10.1111/1348-0421.12687

 

  1. Scurr MJ, Zelek WM, Lippiatt G, et al. Whole blood-based measurement of SARS-CoV-2-specific T cells reveals asymptomatic infection and vaccine immunogenicity in healthy subjects and patients with solid-organ cancers. Immunology. 2022;165(2):250-259. doi: 10.1111/imm.13433

 

  1. Pinto MV, Barkoff AM, Bibi S, et al. A novel whole blood assay to quantify the release of T cell associated cytokines in response to Bordetella pertussis antigens. J Immunol Methods. 2024;534:113758. doi: 10.1016/j.jim.2024.113758

 

  1. Messina NL, Germano S, Chung AW, et al. Effect of bacille calmette-guérin vaccination on immune responses to SARS-CoV-2 and COVID-19 vaccination. Clin Transl Immunology. 2025;14(1):e70023. doi: 10.1002/cti2.70023

 

  1. Antoniak S. The coagulation system in host defense. Res Pract Thromb Haemost. 2018;2(3):549-557. doi: 10.1002/rth2.12109

 

  1. Watanabe S, Zenke K, Muroi M. Lipoteichoic acid inhibits lipopolysaccharide-induced TLR4 signaling by forming an inactive TLR4/MD-2 complex dimer. J Immunol. 2023;210(9):1386-1395. doi: 10.4049/jimmunol.2200872

 

  1. Huldani H, Rashid AI, Turaev KN, et al. Concanavalin A as a promising lectin-based anti-cancer agent: The molecular mechanisms and therapeutic potential. Cell Commun Signal. 2022;20(1):167. doi: 10.1186/s12964-022-00972-7

 

  1. Remick DG, Newcomb DE, Friedland JS. Whole-blood assays for cytokine production. Methods Mol Med. 2000;36:101-112. doi: 10.1385/1-59259-216-3.101

 

  1. Page L, Lauruschkat CD, Dennehy K, et al. Whole blood assay with dual co-stimulation for antigen-specific analysis of host immunity to fungal and viral pathogens. J Vis Exp. 2024;211:e66593. doi: 10.3791/66593

 

  1. Johnson VJ, Luster MI, Edwards A, et al. An in vitro test battery using human whole blood for immunotoxicity hazard identification: Proof of concept studies with dexamethasone and benzo(a)pyrene. Toxicology. 2025;515:154153. doi: 10.1016/j.tox.2025.154153

 

  1. Barclay TG, Day CM, Petrovsky N, Garg S. Review of polysaccharide particle-based functional drug delivery. Carbohydr Polym. 2019;221:94-112. doi: 10.1016/j.carbpol.2019.05.067

 

  1. Buijsers B, Yanginlar C, Maciej-Hulme ML, De Mast Q, Van der Vlag J. Beneficial non-anticoagulant mechanisms underlying heparin treatment of COVID-19 patients. EBioMedicine. 2020;59:102969. doi: 10.1016/j.ebiom.2020.102969

 

  1. Mousavi S, Moradi M, Khorshidahmad T, Motamedi M. Anti-inflammatory effects of heparin and its derivatives: A systematic review. Adv Pharmacol Sci. 2015;2015:507151. doi: 10.1155/2015/507151

 

  1. Makino-Okamura C, Niki Y, Takeuchi S, et al. Heparin inhibits melanosome uptake and inflammatory response coupled with phagocytosis through blocking PI3k/Akt and MEK/ERK signaling pathways in human epidermal keratinocytes. Pigment Cell Melanoma Res. 2014;27(6):1063-1074. doi: 10.1111/pcmr.12287

 

  1. Talsma DT, Katta K, Boersema M, et al. Increased migration of antigen presenting cells to newly-formed lymphatic vessels in transplanted kidneys by glycol-split heparin. PLoS One. 2017;12(6):e0180206. doi: 10.1371/journal.pone.0180206

 

  1. Nguyen KG, Gillam FB, Hopkins JJ, et al. Molecular mechanisms of heparin-induced modulation of human interleukin 12 bioactivity. J Biol Chem. 2019;294(12):4412-4424. doi: 10.1074/jbc.ra118.006193

 

  1. Sato K, Kondo M, Sakuta K, et al. Impact of culture medium on the expansion of T cells for immunotherapy. Cytotherapy. 2009;11(7):936-946. doi: 10.3109/14653240903219114

 

  1. MacPherson S, Keyes S, Kilgour MK, et al. Clinically relevant T cell expansion media activate distinct metabolic programs uncoupled from cellular function. Mol Ther Methods Clin Dev. 2022;24:380-393. doi: 10.1016/j.omtm.2022.02.004

 

  1. Grosick R, Alvarado-Vazquez PA, Messersmith AR, Romero-Sandoval EA. High glucose induces a priming effect in macrophages and exacerbates the production of pro-inflammatory cytokines after a challenge. J Pain Res. 2018;11:1769-1778. doi: 10.2147/Jpr.S164493

 

  1. Witcoski Junior L, De Lima JD, Somensi AG, et al. Metabolic reprogramming of macrophages in the context of type 2 diabetes. Eur J Med Res. 2024;29(1):497. doi: 10.1186/s40001-024-02069-y

 

  1. Mallick S, Duttaroy AK, Bose B. A snapshot of cytokine dynamics: A fine balance between health and disease. J Cell Biochem. 2025;126(1):e30680. doi: 10.1002/jcb.30680

 

  1. Seledtsov VI, Seledtsova GV, Von Delwig AA. Immune-based, multifaceted inactivation of pathogenic T lymphocytes in treating autoimmune diseases. Explor Immunol. 2023;3:506-512. doi: 10.37349/ei.2023.00117

 

  1. Hoyer KK, Dooms H, Barron L, Abbas AK. Interleukin-2 in the development and control of inflammatory disease. Immunol Rev. 2008;226:19-28. doi: 10.1111/j.1600-065X.2008.00697.x

 

  1. Boyman O, Sprent J. The role of interleukin-2 during homeostasis and activation of the immune system. Nat Rev Immunol. 2012;12(3):180-190. doi: 10.1038/nri3156

 

  1. Engelmann B, Massberg S. Thrombosis as an intravascular effector of innate immunity. Nat Rev Immunol. 2013;13(1):34-45. doi: 10.1038/nri3345
Next article in this issue
Share
Back to top
Journal of Biological Methods, Electronic ISSN: 2326-9901 Print ISSN: TBA, Published by POL Scientific
We use cookies on our website to ensure you get the best experience.
Read more about our cookies here
Accept