Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Aug 10;101(32):11737-42.
doi: 10.1073/pnas.0306294101. Epub 2004 Jul 30.

Tapasin enhances MHC class I peptide presentation according to peptide half-life

Mark Howarth  1 , Anthony Williams, Anne B Tolstrup, Tim Elliott
Affiliations

Tapasin enhances MHC class I peptide presentation according to peptide half-life

Mark Howarth et al. Proc Natl Acad Sci U S A. .

Abstract

Understanding how peptides are selected for presentation by MHC class I is crucial to vaccination strategies based on cytotoxic T lymphocyte priming. We have studied this selection of the MHC class I peptide repertoire in terms of the presentation of a series of individual peptides with a wide range of binding to MHC class I. This series was expressed as minigenes, and the presentation of each peptide variant was determined with the same MHC class I peptide-specific antibody. In wild-type cells, the hierarchy of presentation followed peptide half-life. This hierarchy broke down in cells lacking tapasin but not in cells lacking calreticulin or in cells lacking transporter associated with antigen processing-associated ERp57. We demonstrate a key role for tapasin in shaping the MHC class I peptide repertoire, as enhancement of presentation in the presence of tapasin correlated with peptide half-life.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Establishing a hierarchy of peptides based on binding quality. (a) Half-life of Kb:SIINFEKL (♦), SIINFEKV (▴), SIINFEKM (▪), and SIINYEKL (•) complexes and empty molecules (×) on peptide-loaded RMA-S cells is expressed as median fluorescence intensity (MFI) after flow cytometry with Y3. The stabilization produced by the peptide at time 0 is 100%, and 0% is the stabilization in the absence of peptide. (b) Affinity of D1 for Kb–peptide complexes. .220 Kb tapasin cells endogenously expressing the SIINFEKL variants were incubated with D1 for 4 h at 4°C. After one wash in FACS wash, they were resuspended in 200 μl of phycoerythrin-labeled 2° antibody for 30 min at 4°C. After one wash in FACS wash, samples were analyzed by flow cytometry. (c) Binding of SIINFEKL derivatives to Kb at the cell surface of .220Kb measured with Mab 25.D1. (d) Binding of SIINFEKL derivatives to Kb at the cell surface of RMA-S measured with Mab Y3.
Fig. 2.
Fig. 2.
The effect of tapasin on presentation of the hierarchy. Expression level of the SIINFEKL variants in .220Kb-Tpn (a) and .220Kb (b) was determined by flow cytometry using Mab 25.D1. (c) Hierarchy of presentation of the SIINFEKL variants with and without tapasin, expressed as a percentage of SIINFEKL presentation: (peptide MFI – FILKSINE MFI)/(SIINFEKL MFI – FILKSINE MFI) × 100. FILKSINE MFI represents background staining. Means of duplicate measurements are shown ±1 SD. Where error bars are not seen, they are too small to be obvious. (d) Correlation between the enhancement of presentation by tapasin and peptide half-life: enhancement of presentation by tapasin, calculated as (peptide MFI – FILKSINE MFI) with tapasin/(peptide MFI – FILKSINE MFI) without tapasin. Peptide half-life is from Fig. 1a.
Fig. 3.
Fig. 3.
The effect of calreticulin on presentation of the hierarchy. (A) Presentation of the SIINFEKL variants on wild-type fibroblasts was determined by flow cytometry with D1. (B) Presentation of the SIINFEKL variants on calreticulin-knockout fibroblasts was determined by flow cytometry with D1. (C) Hierarchy of presentation with and without calreticulin, expressed as a percentage of SIINFEKL presentation.
Fig. 4.
Fig. 4.
The effect of recruiting ERp57 to the TAP on presentation of the hierarchy. (a) .220Kb(tpn–/–) was transfected with wild-type (WT) or C95A mutant tapasin, and aliquots of cells were incubated with N-ethylmaleimide. Lysates were fractionated by SDS/PAGE and blotted with antitapasin. A band corresponding to disulfide-bonded heterodimers of ERp57 and tapasin was only seen in cells expressing WT tapasin. (b) Peptide presentation on .220Kb, .220Kb-Tpn, or .220Kb C95A tapasin was determined by flow cytometry with D1, and the MFI is displayed. Data for SIINFEKV were not obtained. (c) Hierarchy of presentation with WT or C95A mutant tapasin, expressed as a percentage of SIINFEKL presentation.
Fig. 5.
Fig. 5.
The effect of the T134K mutation on presentation of the hierarchy. (a) The effect of the T134K mutation in Kb on TAP association. 35S-Met/Cys-labeled anti-TAP coimmunoprecipitates (148.3) from .220 Kb tapasin or .220 T134K Kb tapasin cells were released by Triton X-100 and reprecipitated with Y3 (Upper). An aliquot of postnuclear supernatant was immunoblotted for calreticulin as a control for equal cell number (Lower). (b) Level of presentation of the SIINFEKL variants with a T134K mutation in Kb. Peptide presentation on .220 T134K Kb tapasin was determined by flow cytometry with D1. SIINFEKV presentation was not determined. (c) Hierarchy of presentation of the SIINFEKL variants with a T134K mutation in Kb, expressed as a % of SIINFEKL presentation. Means of duplicate measurements are shown ±1 SD.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Shastri, N., Schwab, S. & Serwold, T. (2002) Annu. Rev. Immunol. 20, 463–493. - PubMed
    1. Engelhard, V. H., Brickner, A. G. & Zarling, A. L. (2002) Mol. Immunol. 39, 127–137. - PubMed
    1. Madden, D. R. (1995) Annu. Rev. Immunol. 13, 587–622. - PubMed
    1. Williams, A., Peh, C. A. & Elliott, T. (2002) Tissue Antigens 59, 3–17. - PubMed
    1. Ortmann, B., Copeman, J., Lehner, P. J., Sadasivan, B., Herberg, J. A., Grandea, A. G., Riddell, S. R., Tampe, R., Spies, T., Trowsdale, J. & Cresswell, P. (1997) Science 277, 1306–1309. - PubMed

Publication types

LinkOut - more resources