Abstract
Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.
Highlights
MHC class I (MHC-I) molecules present short (8-10 amino acids) peptides derived from proteins synthesized by each cell
We focus on using the sc MHC class I technology to investigate co-agonism during Hepatitis B virus (HBV) T cell activation, it must be noted that the presented methods can be adapted for research on other aspects of T cell activation in human T cell systems with known pMHC-I specificity
It must be noted that very high percentages of CD107a+ T cells were observed even in response to low levels of antigenic pMHC (Figure 2B), consistent with previous reports of relatively low requirements for the amount of agonist pMHC for induction of T cell cytotoxicity[33]
Summary
MHC class I (MHC-I) molecules present short (8-10 amino acids) peptides derived from proteins synthesized by each cell. Comparison of T cells stimulated with antigen loaded on TAP-sufficient and -deficient mouse RMA and RMA-S cell lines, respectively, as APCs, did not reveal any evidence for co-agonism during mouse T cell activation[18] Use of this experimental system did not allow precise control over the amount of agonist pMHC presented independently of non-stimulatory self pMHC. This was achieved through the following: incubation of TAP2-deficient RMA-S cells at lower temperatures (28 °C, instead of the usual 37 °C) to stabilize empty MHC class I heavy chain/β2 microglobulin complexes[19]; incubation at 28 °C with exogenous agonist peptide; incubation at 28 °C in the presence or absence of exogenous non-stimulatory peptide; and incubation at 37 °C to reduce cell surface expression of empty MHC class I complexes[8,9] Use of this experimental set-up revealed that the presence of non-stimulatory pMHC enhanced activation of mouse thymocytes, naïve peripheral CD8+ T cells and CTLs8. NOTE: All steps involving use of live, non-fixed cells should be performed in a biosafety hood, and any biohazardous waste should be discarded according to the local health and safety regulations
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