Novel in vitro invariant natural killer T cell functional assays

Allison Balasko; Colin Graydon; Keith R Fowke

doi:10.1016/j.jim.2021.113171

Novel in vitro invariant natural killer T cell functional assays

J Immunol Methods. 2021 Dec:499:113171. doi: 10.1016/j.jim.2021.113171. Epub 2021 Oct 24.

Authors

Allison Balasko¹, Colin Graydon², Keith R Fowke³

Affiliations

¹ Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Canada. Electronic address: umbalask@myumanitoba.ca.
² Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Canada.
³ Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Canada; Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya; Department of Community Health Sciences, University of Manitoba, Winnipeg, Canada; Partners for Health and Development in Africa, Nairobi, Kenya. Electronic address: keith.fowke@umanitoba.ca.

PMID: 34706265
DOI: 10.1016/j.jim.2021.113171

Abstract

Background: Invariant Natural Killer T (iNKT) cells are innate lymphocytes bridging the innate and adaptive immune systems and are critical first responders against cancer and infectious diseases. iNKT cell phenotype and functionality are studied using in vitro stimulation assays assessing cytokine response and proliferation capabilities. The most common stimulant is the glycolipid α-Galactosyl Ceramide (α-GalCer), which stimulates iNKT cells when presented by CD1d, an MHC class I-like molecule expressed by antigen-presenting cells (APC). Another stimulant used is α-GalCer-loaded DimerX, a CD1d-Ig fusion protein which stimulates iNKT cells in an APC-independent fashion. Here, we demonstrate use of the PBS-57-loaded CD1d-tetramer as an APC-independent stimulant, where PBS-57 is an α-GalCer analogue.

Methods: Using healthy fresh (n = 4) and frozen (n = 7) peripheral blood mononuclear cells (PBMCs), 10-h cytokine response (measuring IFN-γ production) and 10-day proliferation assays were performed assessing iNKT functionality using α-GalCer, CD1d-tetramer and DimerX stimulants.

Results: All stimulants effectively induced IFN-γ production in both fresh and frozen PBMC. After the 10-h activation, CD1d-tetramer was significantly more effective than α-GalCer (p = 0.032) in inducing IFN-γ production in fresh PBMC and significantly more effective than both α-GalCer (p = 0.004) and DimerX (p = 0.021) in frozen PBMC. Similarly, all stimulants induced strong proliferation responses in all samples, although this was only significant in the frozen PBMC. No significant differences in proliferation were observed between stimulants.

Significance: This study supports PBS-57-loaded CD1d-tetramer as an effective in vitro APC-independent iNKT cell stimulant, which is comparable to or even more effective than α-GalCer and DimerX. As CD1d is downregulated during infectious disease and cancer as evasion strategies, in vitro assays which are APC-independent can assist in providing objective insight to iNKT activation by not relying on CD1d expression by APCs. Overall, the novel CD1d-tetramer stimulation equips researchers with an expanded "toolkit" to successfully assess iNKT cell function.

Keywords: Functional assay; Infectious diseases; Invariant natural killer T cells (iNKT); Tetramer; cancer; α-Galactosyl ceramide (α-GalCer).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Flow Cytometry*
Humans
Natural Killer T-Cells / cytology
Natural Killer T-Cells / immunology*

Grants and funding

319298/CIHR/Canada