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 predetermined 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.

Non-Stimulatory pMHC Enhance CD8 T Cell Effector Functions by Recruiting Coreceptor-Bound Lck

“…We sought to determine if co-agonist pMHC-I complexes can enhance cytokine production in OT-I CD8 + T cells, and expression of molecules implicated in CD8 + T cell effector differentiation. We used the T REX CHO system ( 6 , 26 ) as APCs, which allows presentation of small amounts of agonist OVA peptide on H-2K b in a single chain format (scK b OVA) ( 27 ) in the presence or absence of non-stimulatory VSV on H-2K b , also in single chain format (scK b VSV). As T REX CHO cells do not express any mouse MHC-I molecules, use of this system allows us to compare T cell responses to low amount of agonist pMHC in the presence or absence of non-stimulatory pMHC.…”

“…This was largely due to the technical difficulties in selectively controlling the amounts of agonist and non-stimulatory pMHC under physiological settings. To overcome this problem, we used an adherent xenogeneic cell line expressing agonist or non-stimulatory peptide MHC-I in a single chain format ( 6 , 26 ). Using this system, we confirmed a previous report ( 32 ) that short (4h) antigen stimulation is sufficient for subsequent antigen-independent CD8 + T cell proliferation and effector differentiation in vitro .…”

“…CHO APCs preparation for stimulation experiments was described before ( 26 ). Briefly, one day before the T cell activation experiments, CHO cells were seeded into 96 U-bottom (for flow cytometry analysis) or 12 well flat bottom plates (for imaging flow cytometry analysis and priming for Listeria monocytogenes infection), at 20,000 cells in 100μl or 200,000 cells in 1ml, respectively.…”

Targeting CAR to the Peptide-MHC Complex Reveals Distinct Signaling Compared to That of TCR in a Jurkat T Cell Model

“…Peptide mutagenesis: GAG (SLYNTVATL) to LMP2 A426-434 ("L2": CLGGLLTMV) was done by using a Q5 mutagenesis Kit (New England Biolabs, Ipswich, MA, USA). All the molecular cloning work was done by using an In-Fusion HD cloning kit (Clontech, Mountain View, CA, USA), and single-chain trimer MHC constructs were cloned into pLv to generate artificial antigen-presenting CHO cells [21][22][23].…”

“…This was performed largely as described [22,23]. Artificial antigen presenting CHO cells (CHO-APC) were seeded onto a 96-well, flat-bottom plate at 2-3 × 10 4 per well one day before the assay.…”