Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Canada.
Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Canada.
J Immunol Methods. 2021 Dec;499:113171. doi: 10.1016/j.jim.2021.113171. Epub 2021 Oct 24.
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.
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.
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.
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.
不变自然杀伤 T(iNKT)细胞是连接先天和适应性免疫系统的先天淋巴细胞，是对抗癌症和传染病的关键早期反应细胞。通过评估细胞因子反应和增殖能力的体外刺激试验来研究 iNKT 细胞的表型和功能。最常用的刺激物是糖脂α-半乳糖基神经酰胺（α-GalCer），当由抗原呈递细胞（APC）表达的 MHC 类 I 样分子 CD1d 呈递时，它会刺激 iNKT 细胞。另一种常用的刺激物是负载α-GalCer 的二聚体 X（DimerX），它是一种 CD1d-Ig 融合蛋白，以 APC 非依赖性的方式刺激 iNKT 细胞。在这里，我们证明了负载 PBS-57 的 CD1d 四聚体作为 APC 非依赖性刺激物的用途，其中 PBS-57 是一种α-GalCer 类似物。
使用健康的新鲜（n=4）和冷冻（n=7）外周血单核细胞（PBMC），进行 10 小时细胞因子反应（测量 IFN-γ 产生）和 10 天增殖试验，使用 α-GalCer、CD1d 四聚体和 DimerX 刺激物评估 iNKT 功能。
所有刺激物均能有效诱导新鲜和冷冻 PBMC 产生 IFN-γ。在 10 小时激活后，CD1d 四聚体在新鲜 PBMC 中诱导 IFN-γ产生的效果明显优于α-GalCer（p=0.032），在冷冻 PBMC 中诱导 IFN-γ产生的效果明显优于α-GalCer（p=0.004）和 DimerX（p=0.021）。同样，所有刺激物在所有样本中均诱导强烈的增殖反应，尽管在冷冻 PBMC 中仅观察到显著的增殖反应。在刺激物之间未观察到增殖的显著差异。
这项研究支持负载 PBS-57 的 CD1d 四聚体作为有效的体外 APC 非依赖性 iNKT 细胞刺激物，其与α-GalCer 和 DimerX 相当或甚至更有效。由于 CD1d 在传染病和癌症期间作为逃避策略而下调，因此非 APC 依赖性的体外试验可以通过不依赖 APC 表达 CD1d 来帮助提供对 iNKT 激活的客观见解。总体而言，新型 CD1d 四聚体刺激为研究人员提供了扩展的“工具包”，以成功评估 iNKT 细胞功能。
