Reversible major histocompatibility complex I-peptide multimers containing Ni(2+)-nitrilotriacetic acid peptides and histidine tags improve analysis and sorting of CD8(+) T cells.

MHC-peptide multimers containing biotinylated MHC-peptide complexes bound to phycoerythrin (PE) streptavidin (SA) are widely used for analyzing and sorting antigen-specific T cells. Here we describe alternative T cell-staining reagents that are superior to conventional reagents. They are built on reversible chelate complexes of Ni(2+)-nitrilotriacetic acid (NTA) with oligohistidines. We synthesized biotinylated linear mono-, di-, and tetra-NTA compounds using conventional solid phase peptide chemistry and studied their interaction with HLA-A*0201-peptide complexes containing a His(6), His(12), or 2×His(6) tag by surface plasmon resonance on SA-coated sensor chips and equilibrium dialysis. The binding avidity increased in the order His(6) < His(12) < 2×His(6) and NTA(1) < NTA(2) < NTA(4), respectively, depending on the configuration of the NTA moieties and increased to picomolar K(D) for the combination of a 2×His(6) tag and a 2×Ni(2+)-NTA(2). We demonstrate that HLA-A2-2×His(6)-peptide multimers containing either Ni(2+)-NTA(4)-biotin and PE-SA- or PE-NTA(4)-stained influenza and Melan A-specific CD8+ T cells equal or better than conventional multimers. Although these complexes were highly stable, they very rapidly dissociated in the presence of imidazole, which allowed sorting of bona fide antigen-specific CD8+ T cells without inducing T cell death as well as assessment of HLA-A2-peptide monomer dissociation kinetics on CD8+ T cells.

Antigen-containing liposomes engrafted with flagellin-related peptides are effective vaccines that can induce potent antitumor immunity and immunotherapeutic effect.

The bacterial protein flagellin can trigger immune responses to infections by interacting with TLR5 on APCs, and Ag-flagellin fusion proteins can act as effective vaccines. We report that flagellin-related peptides containing a His-tag and sequence related to conserved N-motif (aa 85-111) of FliC flagellin, purportedly involved in the interaction of flagellin with TLR5, can be used to target delivery of liposomal Ag to APCs in vitro and in vivo. When engrafted onto liposomes, two flagellin-related peptides, denoted as 9Flg and 42Flg, promoted strong liposome binding to murine bone marrow-derived dendritic cells and CD11c(+) splenocytes, and cell binding correlated with expression of TLR5. Liposomes engrafted with 9Flg or 42Flg induced functional MyD88-dependent maturation of dendritic cells in vivo. The vaccination of mice with 9Flg liposomes containing OVA induced OVA-specific T cell priming, increased the number of Ag-responsive IFN-gamma-producing CD8(+) T cells, and increased Ag-specific IgG(1) and IgG(2b) in serum. Importantly, the vaccination of C57BL/6 mice with syngeneic B16-OVA-derived plasma membrane vesicles, engrafted with 9Flg or 42Flg, potently inhibited tumor growth/metastasis and induced complete tumor regression in the majority of mice challenged with the syngeneic B16-OVA melanoma, in the lung and s.c. tumor models. Strong antitumor responses were also seen in studies using the s.c. P815 tumor model. Therefore, vaccination with Ag-containing liposomes engrafted with 9Flg or 42Flg is a powerful strategy to exploit the innate and adaptive immune systems for the development of potent vaccines and cancer immunotherapies.