Structural Engineering of pMHC Reagents for T Cell Vaccines and Diagnostics

Mitaksov, Vesselin; Truscott, Steven M.; Lybarger, Lonnie; Connolly, Jennifer M.; Hansen, Ted H.; Fremont, Daved H.

doi:10.1016/j.chembiol.2007.07.010

Cited by 56 publications

(80 citation statements)

References 59 publications

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“…The SCT-K b construct was modified to produce a more stable SCT-K b in which the SIINFEKL peptide sequence was covalently linked to the α1 helix of K b (25,26). Tetramers of this SCT-K b molecule bound specifically to Ly49C + NK cells.…”

Section: Resultsmentioning

confidence: 99%

Natural killer cell licensing in mice with inducible expression of MHC class I

Ebihara

Jonsson

Yokoyama

2013

Proc. Natl. Acad. Sci. U.S.A.

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Mouse natural killer (NK) cells acquire effector function by an education process termed "licensing" mediated by inhibitory Ly49 receptors which recognize self-MHC class I. Ly49 receptors can bind to MHC class I on targets (in trans) and also to MHC class I on the NK-cell surface (in cis). Which of these interactions regulates NKcell licensing is not yet clear. Moreover, there are no clear phenotypic differences between licensed and unlicensed NK cells, perhaps because of the previously limited ability to study NK cells with synchronized licensing. Here, we produced MHC class I-deficient mice with inducible MHC class I consisting of a single-chain trimer (SCT), ovalbumin peptide-β2 microgloblin-H2K b (SCT-K b ). Only NK cells with a Ly49 receptor with specificity for SCT-K b were licensed after MHC class I induction. NK cells were localized consistently in red pulp of the spleen during induced NK-cell licensing, and there were no differences in maturation or activation markers on recently licensed NK cells. Although MHC class I-deficient NK cells were licensed in hosts following SCT-K b induction, NK cells were not licensed after induced SCT-K b expression on NK cells themselves in MHC class I-deficient hosts. Furthermore, hematopoietic cells with induced SCT-K b licensed NK cells more efficiently than stromal cells. These data indicate that trans interaction with MHC class I on hematopoietic cells regulates NK-cell licensing, which is not associated with other obvious phenotypic changes.tolerance | immunity | lymphocytes

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Section: Resultsmentioning

confidence: 99%

Natural killer cell licensing in mice with inducible expression of MHC class I

Ebihara

Jonsson

Yokoyama

2013

Proc. Natl. Acad. Sci. U.S.A.

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“…In this regard, we have successfully incorporated a disulfide trap into recombinant K b /OVA without SCT linkers by introducing cysteines into the recombinant heavy chain protein and synthetic peptide. These disulfide trap MHC-I tetramers stained T cells specific for K b /OVA as well as NK cells expressing the cognate Ly49 NK inhibitory receptor (24). Thus the disulfide trap preserves MHC-I antigen specificity and is a promising option where the loss of peptide is a critical limitation.…”

Section: Discussionmentioning

confidence: 99%

“…The clear implication of these findings was that the superior surface stability of the SCT compared with native K b /OVA was not due to continuous peptide binding, but rather the ability to rebind the linker-attached peptide. Subsequent crystallographic studies of the K b /OVA SCT demonstrated that the linker extending from the OVA peptide moiety disrupted a highly conserved H-bonding network that normally serves to anchor peptide at the C terminus (24). Specifically, the heavy chain residue Tyr-84 forced the linker to protrude above from the peptide binding platform, thereby disrupting the H-bonding network of the peptide C terminus with the heavy chain.…”

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confidence: 99%

“…To better accommodate linker extension and also improve C-terminal peptide anchoring, we recently engineered a disulfide trap into murine SCT molecules (dtSCTs) between position 84 of the MHC-I heavy chain and the second position on the SCT linker extending from the C terminus of the peptide (24,25). Not only did the dtSCT constructs fold efficiently in cells, but the disulfide trap oxidized in a reliable manner and displayed a tenacious ability to prevent the binding of exogenous peptides.…”

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confidence: 99%

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Human Major Histocompatibility Complex (MHC) Class I Molecules with Disulfide Traps Secure Disease-related Antigenic Peptides and Exclude Competitor Peptides

Truscott

Wang

Lybarger

et al. 2008

Journal of Biological Chemistry

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The ongoing discovery of disease-associated epitopes detected by CD8 T cells greatly facilitates peptide-based vaccine approaches and the construction of multimeric soluble recombinant proteins (e.g. tetramers) for isolation and enumeration of antigen-specific CD8 T cells. Related to these outcomes of epitope discovery is the recent demonstration that MHC class I/peptide complexes can be expressed as single chain trimers (SCTs) with peptide, ␤ 2 m and heavy chain connected by linkers to form a single polypeptide chain. Studies using clinically relevant mouse models of human disease have shown that SCTs expressed by DNA vaccination are potent stimulators of cytotoxic T lymphocytes. Their vaccine efficacy has been attributed to the fact that SCTs contain a preprocessed and preloaded peptide that is stably displayed on the cell surface. Although SCTs of HLA class I/peptide complexes have been previously reported, they have not been characterized for biochemical stability or susceptibility to exogenous peptide binding. Here we demonstrate that human SCTs remain almost exclusively intact when expressed in cells and can incorporate a disulfide trap that dramatically excludes the binding of exogenous peptides. The mechanistic and practical applications of these findings for vaccine development and T cell isolation/enumeration are discussed.

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“…Importantly, SCTs allow peptides with very weak affinity for MHC to be anchored into the peptide binding groove through a disulfide bond between the linker and heavy chain ( Fig. 2A) (7)(8)(9). Moreover, SCTs are recognized as intact molecules, because the D227K mutation in the α3 domain of the OVA/K b SCT abrogates CD8 interaction (14) and induces diminished K b -specific CD8 + T-cell responses when used in a DNA vaccine (Fig.…”

Section: Resultsmentioning

confidence: 99%

Cross-dressed CD8α ⁺ /CD103 ⁺ dendritic cells prime CD8 ⁺ T cells following vaccination

Li¹,

Kim

Herndon³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Activation of naïve cluster of differentiation (CD)8 + cytotoxic T lymphocytes (CTLs) is a tightly regulated process, and specific dendritic cell (DC) subsets are typically required to activate naive CTLs. Potential pathways for antigen presentation leading to CD8 + T-cell priming include direct presentation, cross-presentation, and cross-dressing. To distinguish between these pathways, we designed single-chain trimer (SCT) peptide-MHC class I complexes that can be recognized as intact molecules but cannot deliver antigen to MHC through conventional antigen processing. We demonstrate that cross-dressing is a robust pathway of antigen presentation following vaccination, capable of efficiently activating both naïve and memory CD8 + T cells and requires CD8α + / CD103 + DCs. Significantly, immune responses induced exclusively by cross-dressing were as strong as those induced exclusively through cross-presentation. Thus, cross-dressing is an important pathway of antigen presentation, with important implications for the study of CD8 + T-cell responses to viral infection, tumors, and vaccines. P rofessional antigen-presenting cells (APCs) are typically required to activate naïve cluster of differentiation (CD)8 + T cells, either by direct priming or cross-priming. In direct priming, infected (viral infection) or directly transfected (DNA vaccination) APCs synthesize the foreign antigen and use endogenous MHC class I pathways of antigen presentation to present antigen and prime CD8 + T cells. In cross-priming, APCs are able to capture, process, and present exogenous antigen onto MHC class I molecules through a process known as cross-presentation (1). Cross-priming has been shown to be an essential pathway for immunity to many viral infections and tumors. Although the pathways that lead to cross-presentation remain incompletely understood, increasing evidence suggests that only certain dendritic cell (DC) subsets are efficient in this process.Cross-dressing involves the transfer of intact MHC class I/ peptide complexes between cells without the requirement for further processing, representing an alternative pathway of indirect antigen presentation (2, 3). Although cross-dressed DCs can activate memory CD8 + T cells following viral infection in vivo (4), it remains unclear whether cross-dressing can prime naïve CD8 + T-cell responses, what DC subtypes are required to prime CD8 + T cells by cross-dressing, and how robust this pathway is compared with traditional pathways of indirect antigen presentation. These questions must be addressed before the physiologic relevance of cross-dressing can be evaluated in context.To address these questions, we have taken advantage of Batf3-deficient mice and engineered MHC class I single chain trimer (SCT) constructs. Batf3 −/− mice have a selective loss of CD8α + and CD103 + DCs, without abnormalities in other hematopoietic cell types or architecture (5). DCs from Batf3 −/− mice are deficient in cross-presentation, and cytotoxic T lymphocyte (CTL) responses to viral infection and synge...

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Structural Engineering of pMHC Reagents for T Cell Vaccines and Diagnostics

Cited by 56 publications

References 59 publications

Natural killer cell licensing in mice with inducible expression of MHC class I

Natural killer cell licensing in mice with inducible expression of MHC class I

Human Major Histocompatibility Complex (MHC) Class I Molecules with Disulfide Traps Secure Disease-related Antigenic Peptides and Exclude Competitor Peptides

Cross-dressed CD8α ⁺ /CD103 ⁺ dendritic cells prime CD8 ⁺ T cells following vaccination

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Structural Engineering of pMHC Reagents for T Cell Vaccines and Diagnostics

Cited by 56 publications

References 59 publications

Natural killer cell licensing in mice with inducible expression of MHC class I

Natural killer cell licensing in mice with inducible expression of MHC class I

Human Major Histocompatibility Complex (MHC) Class I Molecules with Disulfide Traps Secure Disease-related Antigenic Peptides and Exclude Competitor Peptides

Cross-dressed CD8α + /CD103 + dendritic cells prime CD8 + T cells following vaccination

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Cross-dressed CD8α ⁺ /CD103 ⁺ dendritic cells prime CD8 ⁺ T cells following vaccination