Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

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.

Time required for commitment to T cell proliferation depends on TCR affinity and cytokine response

“…Single chain pMHCs for H2‐K b (scK b ) with peptides of different affinity to OT‐I TCR were expressed under the control of doxycycline‐inducible promoter in the hamster‐derived T‐REx CHO cell line expressing doxycycline repressor. This allows very low expression of pMHC when doxycycline is not added (Hoerter et al , 2013; Zhao et al , 2018, 2019). We used scK b ‐OVA, Q4R7, Q4H7, Q7, G4 and VSV as a set of pMHC with gradually decreasing affinity to stimulate OT‐I T cells (Hogquist et al , 1994; Alam et al , 1996; Rosette et al , 2001; Daniels et al , 2006; Stepanek et al , 2014) (Fig EV1).…”

“…Different single chain (sc) pMHC (SIINFEKL (OVA), SIIQFERL (Q4R7), SIIQFEHL (Q4H7), SIINFEQL (Q7), SIIGFEKL (G4), or RGYVYQGL (VSV)) were expressed under the control of a tetracycline‐inducible promoter in T‐REx CHO cells expressing tetracycline repressor (Hoerter et al , 2013; Zhao et al , 2019). The cells were single cell sorted, and clones with matching H‐2Kb expression levels were used as APC in T cell stimulation assays (Hoerter et al , 2013; Zhao et al , 2018).…”

“…One day prior to T cell activation, T‐Rex CHO cells expressing sc‐pMHC were treated with 0.05 ng/ml mitomycin C for 3 h and washed with cRPMI twice (Zhao et al , 2019). Mitomycin C was used to abolish CHO cell proliferation without damaging APC function.…”