Soluble MHC-Peptide Complexes Containing Long Rigid Linkers Abolish CTL-Mediated Cytotoxicity

Abstract

Soluble MHC-peptide (pMHC) complexes induce intracellular calcium mobilization, diverse phosphorylation events, and death of CD8+ CTL, given that they are at least dimeric and coengage CD8. By testing dimeric, tetrameric, and octameric pMHC complexes containing spacers of different lengths, we show that their ability to activate CTL decreases as the distance between their subunit MHC complexes increases. Remarkably, pMHC complexes containing long rigid polyproline spacers (≥80 Å) inhibit target cell killing by cloned S14 CTL in a dose- and valence-dependent manner. Long octameric pMHC complexes abolished target cell lysis, even very strong lysis, at nanomolar concentrations. By contrast, an altered peptide ligand antagonist was only weakly inhibitory and only at high concentrations. Long Db-gp33 complexes strongly and specifically inhibited the Db-restricted lymphocytic choriomeningitis virus CTL response in vitro and in vivo. We show that complications related to transfer of peptide from soluble to cell-associated MHC molecules can be circumvented by using covalent pMHC complexes. Long pMHC complexes efficiently inhibited CTL target cell conjugate formation by interfering with TCR-mediated activation of LFA-1. Such reagents provide a new and powerful means to inhibit Ag-specific CTL responses and hence should be useful to blunt autoimmune disorders such as diabetes type I.

Increased Mobility of Major Histocompatibility Complex I-Peptide Complexes Decreases the Sensitivity of Antigen Recognition

“…Antigen recognition by S14 CTL was assessed by 51 Cr release experiments as described (33). Conjugate formation was assessed as described previously (37). In brief, Indo-1-labeled S14 CTL were mixed with carboxyfluorescein succinimidyl ester (CFSE)-labeled L-K d cells previously sensitized with graded concentrations of peptide 1 (CTL/target ϭ 1 : 1), centrifuged for 1 min at 1000 ϫ g. After 1 min incubation at 37°C CFSE and Indo-1 cell-associated fluorescence were analyzed by flow cytometry on an LSR flow cytometer (BD Biosciences).…”

Active Antiviral T-Lymphocyte Response Can Be Redirected against Tumor Cells by Antitumor Antibody × MHC/Viral Peptide Conjugates

“…To prevent peptide dissociation from the MHCbinding groove in vitro and in vivo, covalent H-2D b /GP33 peptide MHC complexes were prepared by replacing the Lys 33 of the GP33-41 peptide (KAVYNFATA), by a photoreactive 4-azidophenylalanine [F(N3)-AVYNFATA, thereafter referred as cross-linked peptide]. The purified refolded complexes were then UV irradiated at 312 nm for 40 s to photo-crosslink the peptide in the MHC-binding groove (34). To test the recognition of the photo-crosslinked complexes by H-2D b / GP33 -specific CD8 + T cells, H-2D b /GP33 tetramers were made with the cross-linked peptide and compared with wild-type peptide tetramers for the staining of peripheral blood mononuclear cells from LCMV-infected mice.…”

Reversible Major Histocompatibility Complex I-Peptide Multimers Containing Ni2+-Nitrilotriacetic Acid Peptides and Histidine Tags Improve Analysis and Sorting of CD8+ T Cells

“…Because we have shown that the induced dissociation of DTB multimers from CTL is sufficiently rapid to prevent intracellular Ca 2ϩ mobilization (4), the same is expected for NTA-His tag multimers. Intracellular Ca 2ϩ mobilization is required for most CTL functions, including multimer-induced CD8ϩ T cell death (8,26,33).…”