| 1. |
- Geironson Ulfsson, Linda
(författare)
-
HLA class I maturation - in the presence and absence of tapasin
- 2012
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Human leukocyte antigen class I (HLA-I) molecules are present on all nucleated cells and present the cell content to cytotoxic T lymphocytes in the form of peptides. Maturation of HLA-I occurs in the endoplasmic reticulum and results in stable peptide-HLA-I complexes, in the presence of proper quality control. For proper peptide binding most HLA-I molecules interact with the peptide-loading complex (PLC), which is a multi-protein complex consisting of the transporter associated with antigen presentation, calreticulin, ERp57 and tapasin. Tapasin integrates HLA-I into the PLC and mediates quality control of the HLA-I maturation. When an optimal peptide is bound, tapasin releases the peptide-loaded HLA-I molecule that is next transported to the cell surface. The mechanisms for tapasin quality control of HLA-I maturation and the criteria defining optimal peptides are not completely known. Here, a recombinant part of tapasin, the first 87 N-terminal amino acids (Tpn1-87), was produced and shown to facilitate folding of different HLA-I molecules, i.e. allomorphs, to different degree in a peptide dependent manner. Folding of HLA-A*02:01 molecules with natural ligands, i.e. with peptides purified mainly from HLA-I expressed on the cell surface, was not facilitated by Tpn1-87, while folding of non-natural ligands, i.e. not presented at the cell surface, was facilitated. The folding facilitation exerted by Tpn1-87, tapasin-facilitation, inversely correlated with the stability of the peptide-HLA-I complex to some extent. The inverse correlation of these two parameters, tapasin-facilitation and stability, was studied in detail in the third paper. An increased stability was shown to not necessarily be associated with a decreased tapasin-facilitation. In the last paper of this thesis the tapasin-facilitation was studied in a large set of allomorphs folded with peptides of 7 to 13 amino acids in length. The influence of peptide-length for the different allomorphs increased with their tapasin dependence. In conclusion, Tpn1-87 facilitates folding of HLA-I in a peptide- and allomorph-dependent manner. Based on the above studies and data showing tapasin retention of immature HLA-I molecules, and studies of a suggested peptide-editing role of tapasin, we propose that tapasin keeps HLA-I molecules in a peptide-receptive conformation, which reduces the risk degradation of HLA-I molecules and increases the possibility for binding of optimal peptides.
|
|
| 2. |
- Geironson Ulfsson, Linda, et al.
(författare)
-
Stability of peptide-HLA-I complexes and tapasin folding facilitation - tools to define immunogenic peptides
- 2012
-
Ingår i: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 586:9, s. 1336-1343
-
Tidskriftsartikel (refereegranskat)abstract
- Only a small fraction of the peptides generated inside the cell end up being presented by HLA-I on the cell surface. High stability of peptide-HLA-I complexes and a low HLA-I tapasin-facilitation have been proposed to predict immunogenicity. We here set out to investigate if these parameters correlated and defined immunogenic peptides. Both peptide-HLA-B*08:01 and peptide-HLA-A*02:01 complexes showed small differences in tapasin-facilitation and larger differences in stability. This suggests that the stability of immunogenic peptide-HLA-I complexes vary above an HLA-I allomorph dependent lower limit (e. g. > 2 h for HLA-A*02:01), immunogenicity predicted by tapasin-facilitation may be defined by an equally allomorph unique upper value (e. g. tapasin-facilitation <1.5 for HLA-A*02:01), and variation above the stability-threshold does not directly reflect a variation in tapasin-facilitation. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
|
|
| 3. |
|
|
| 4. |
- Geironson Ulfsson, Linda, et al.
(författare)
-
Tapasin facilitation of MHC-I separates closely related allomorphs, is strongly influenced by peptide length and depends on stability
- 2013
-
Ingår i: ; , s. 83-83
-
Konferensbidrag (refereegranskat)abstract
- Only a small fraction of the peptides inside a cell are eventually presented by HLA-I on the cell surface. The presented peptides have HLA-I allomorph-specific motifs and length restrictions. Tapasin influences HLA-I antigen presentation both qualitatively and quantitatively to different degrees depending on both peptide sequence and HLA-I allomorph. The tapasin-dependence in cellular context has been shown to correspond to the facilitation of peptide- HLA-I complex formation by the first 87 amino acids of tapasin (Tpn1- 87) (i.e., tapasin-facilitation = Bmax Tpn1-87/Bmax Ctrl) in a biochemical assay. Both peptide length and tapasin-facilitation are important for HLA-I antigen presentation and we here set out to study if these two parameters relate to each other. We used a luminescent oxygen channeling assay and seven different peptide libraries (X7- X13) to study 16 HLA-A and -B allomorphs and the results show a broad spectrum of tapasin-facilitation of HLA-I allomorphs and that HLA-A allomorphs were generally less restricted than -B allomorphs83to peptides of the classical lengths of 8-10 amino acids. Since both stability and tapasin-facilitation have been suggested as discriminators of immunogenic peptides we used a scintillation proximity based assay to study the stability of peptide-HLA-I complexes formed with peptides of different lengths. The results demonstrate an inverse correlation between tapasin-facilitation and stability valid for different peptide mixes of specific lengths but also on the level of HLA-I allomorphs, suggesting that molecules of poor stability are either not in a conformation that allows tapasin to interact or have a conformation where association has no effect.
|
|
| 5. |
- Geironson Ulfsson, Linda, et al.
(författare)
-
Tapasin Facilitation of Natural HLA-A and -B Allomorphs Is Strongly Influenced by Peptide Length, Depends on Stability, and Separates Closely Related Allomorphs.
- 2013
-
Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 1550-6606 .- 0022-1767. ; 191:7, s. 3939-3947
-
Tidskriftsartikel (refereegranskat)abstract
- Despite an abundance of peptides inside a cell, only a small fraction is ultimately presented by HLA-I on the cell surface. The presented peptides have HLA-I allomorph-specific motifs and are restricted in length. So far, detailed length studies have been limited to few allomorphs. Peptide-HLA-I (pHLA-I) complexes of different allomorphs are qualitatively and quantitatively influenced by tapasin to different degrees, but again, its effect has only been investigated for a small number of HLA-I allomorphs. Although both peptide length and tapasin dependence are known to be important for HLA-I peptide presentation, the relationship between them has never been studied. In this study, we used random peptide libraries from 7- to 13-mers and studied binding in the presence and absence of a recombinant truncated form of tapasin. The data show that HLA-I allomorphs are differentially affected by tapasin, different lengths of peptides generated different amounts of pHLA-I complexes, and HLA-A allomorphs are generally less restricted than HLA-B allomorphs to peptides of the classical length of 8-10 aa. We also demonstrate that tapasin facilitation varies for different peptide lengths, and that the correlation between high degree of tapasin facilitation and low stability is valid for different random peptide mixes of specific lengths. In conclusion, these data show that tapasin has specificity for the combination of peptide length and HLA-I allomorph, and suggest that tapasin promotes formation of pHLA-I complexes with high on and off rates, an important intermediary step in the HLA-I maturation process.
|
|
| 6. |
|
|
| 7. |
- Roder, Gustav, et al.
(författare)
-
Tapasin discriminates peptide-human leukocyte antigen-A*02:01 complexes fromed with natural ligands
- 2011
-
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 286:23, s. 20547-20557
-
Tidskriftsartikel (refereegranskat)abstract
- A plethora of peptides are generated intracellularly, and most peptide-human leukocyte antigen (HLA)-I interactions are of a transient, unproductive nature. Without a quality control mechanism, the HLA-I system would be stressed by futile attempts to present peptides not sufficient for the stable peptide-HLA-I complex formation required for long term presentation. Tapasin is thought to be central to this essential quality control, but the underlying mechanisms remain unknown. Here, we report that the N-terminal region of tapasin, Tpn(1-87), assisted folding of peptide-HLA-A*02:01 complexes according to the identity of the peptide. The facilitation was also specific for the identity of the HLA-I heavy chain, where it correlated to established tapasin dependence hierarchies. Two large sets of HLA-A*02:01 binding peptides, one extracted from natural HLA-I ligands from the SYFPEITHI database and one consisting of medium to high affinity non-SYFPEITHI ligands, were studied in the context of HLA-A*02:01 binding and stability. We show that the SYFPEITHI peptides induced more stable HLA-A*02:01 molecules than the other ligands, although affinities were similar. Remarkably, Tpn(1-87) could functionally discriminate the selected SYFPEITHI peptides from the other peptide binders with high sensitivity and specificity. We suggest that this HLA-I- and peptide-specific function, together with the functions exerted by the more C-terminal parts of tapasin, are major features of tapasin-mediated HLA-I quality control. These findings are important for understanding the biogenesis of HLA-I molecules, the selection of presented T-cell epitopes, and the identification of immunogenic targets in both basic research and vaccine design.
|
|
| 8. |
- Roder, Gustav, et al.
(författare)
-
The outermost N-terminal region of tapasin facilitates folding of major histocompatibility complex class I
- 2009
-
Ingår i: European Journal of Immunology. - : Wiley. - 1521-4141 .- 0014-2980. ; 39:10, s. 2682-2694
-
Tidskriftsartikel (refereegranskat)abstract
- Tapasin (Tpn) is an ER chaperone that is uniquely dedicated to MHC-I biosynthesis. It binds MHC-I molecules, integrates them into peptide-loading complexes, and exerts quality control of the bound peptides; only when an "optimal peptide" is bound will the MHC-I be released and exported to the cell surface for presentation to T cells. The exact mechanisms of Tpn quality control and the criteria for being an optimal peptide are still unknown. Here, we have generated a recombinant fragment of human Tpn, Tpn(1-87) (representing the 87 N-terminal and ER-luminal amino acids of the mature Tpn protein). Using a biochemical peptide-MHC-I-binding assay, recombinant Tpn(1-87) was found to specifically facilitate peptide-dependent folding of HLA-A*0201. Furthermore, we used Tpn(1-87) to generate a monoclonal antibody, alpha Tpn(1-87/80), specific for natural human Tpn and capable of cellular staining of ER localized Tpn. Using overlapping peptides, the epitope of alpha Tpn(1-87)/80 was located to Tpn(40-44), which maps to a surface-exposed loop on the Tpn structure. Together, these results demonstrate that the N-terminal region of Tpn can be recombinantly expressed and adopt a structure, which at least partially resembles that of WT Tpn, and that this region of Tpn features chaperone activity facilitating peptide binding of MHC-I.
|
|
| 9. |
- Røder, Gustav, et al.
(författare)
-
MHC Class I Quality Control
- 2012
-
Ingår i: Histocompatibility. - 9789535105893
-
Bokkapitel (refereegranskat)
|
|
| 10. |
|
|
| 11. |
|
|
| 12. |
- Safi, Fatemeh, et al.
(författare)
-
In vitro clonal multilineage differentiation of distinct murine hematopoietic progenitor populations
- 2023
-
Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 4:1
-
Tidskriftsartikel (refereegranskat)abstract
- Here we describe an in vitro co-culture system that can differentiate hematopoietic progenitor populations to all major hematopoietic lineages at clonal level. We present both a sensitive single-cell switch-culture system as well as a less laborious alternative barcoding protocol more convenient for larger cell numbers. Importantly, generation of all lineages from single long-term hematopoietic stem cells are described, following 21 days of culture. This protocol represents an efficient tool for validation experiments for single-cell genomics data. For complete details on the use and execution of this protocol, please refer to Safi et al. (2022).1
|
|
| 13. |
|
|
| 14. |
|
|
| 15. |
- Thuring, Camilla, et al.
(författare)
-
HLA class I is most tightly linked to levels of tapasin compared with other antigen-processing proteins in glioblastoma
- 2015
-
Ingår i: British Journal of Cancer. - : Springer Science and Business Media LLC. - 1532-1827 .- 0007-0920. ; 113:6, s. 952-962
-
Tidskriftsartikel (refereegranskat)abstract
- Tumour cells can evade the immune system by dysregulation of human leukocyte antigens (HLA-I). Low quantity and/or altered quality of HLA-I cell surface expression is the result of either HLA-I alterations or dysregulations of proteins of the antigen-processing machinery (APM). Tapasin is an APM protein dedicated to the maturation of HLA-I and dysregulation of tapasin has been linked to higher malignancy in several different tumours.
|
|
| 16. |
- Thuring, Camilla, et al.
(författare)
-
Tapasin and human leukocyte antigen class I dysregulation correlates with survival in glioblastoma multiforme.
- 2014
-
Ingår i: Anti-Cancer Agents in Medicinal Chemistry. - 1875-5992. ; 14:8, s. 1101-1109
-
Tidskriftsartikel (refereegranskat)abstract
- Human leukocyte antigen class I (HLA-I) molecules present antigenic peptides to cytotoxic CD8(+) T cells. Downregulation of peptide:HLA-I complexes is common in tumors and results in tumor immune escape variants. Also molecules involved in the maturation of HLA-I have been demonstrated to be dysregulated in malignant neoplasms. We here set out to investigate the antigen presentation capabilities of a set of 12 glioblastoma multiforme (GBM) tumors based on the expression of HLA-I. Moreover, we analyzed the expression of tapasin, a protein dedicated and essential to HLA-I maturation, as well as the infiltration of CD8+ cells using immunohistochemistry on paraffin-embedded sections. Comparison of different GBMs showed a variation in expression of both HLA-I heavy chain (HC) and tapasin. Interestingly, the expression of tapasin and HLA-I HC correlated significantly (p=0.0002) suggesting tapasin to be a key factor for efficient HLA-I antigen presentation in GBMs. Although no statistically significant correlation between CD8(+) cells and survival was found, probably due to a very low number of infiltrating CD8(+) cells at the time of surgical resection, both tapasin and HLA-I HC levels significantly correlated with survival. We suggest that analysis of expression of tapasin and/or HLA-I may be of value as prognostic tool for GBM patients, especially when considering immunotherapy.
|
|
| 17. |
|
|
| 18. |
- Warfvinge, Rebecca, et al.
(författare)
-
Single-cell molecular analysis defines therapy response and immunophenotype of stem cell subpopulations in CML
- 2017
-
Ingår i: Blood. - : AMER SOC HEMATOLOGY. - 0006-4971 .- 1528-0020. ; 129:17, s. 2384-2394
-
Tidskriftsartikel (refereegranskat)abstract
- Understanding leukemia heterogeneity is critical for the development of curative treatments as the failure to eliminate therapy-persistent leukemic stem cells (LSCs) may result in disease relapse. Here we have combined high-throughput immunopheno-typic screens with large-scale single-cell gene expression analysis to define the heterogeneity within the LSC population in chronic phase chronic myeloid leukemia (CML) patients at diagnosis and following conventional tyrosine kinase inhibitor (TKI) treatment. Our results reveal substantial heterogeneity within the putative LSC population in CML at diagnosis and demonstrate differences in response to subsequent TKI treatment between distinct subpopulations. Importantly, LSC subpopulations with myeloid and proliferative molecular signatures are proportionally reduced at a higher extent in response to TKI therapy compared with subfractions displaying primitive and quiescent signatures. Additionally, cell surface expression of the CML stem cell markers CD25, CD26, and IL1RAP is high in all subpopulations at diagnosis but downregulated and unevenly distributed across subpopulations in response to TKI treatment. The most TKI-insensitive cells of the LSC compartment can be captured within the CD45RA(-) fraction and further defined as positive for CD26 in combination with an aberrant lack of cKIT expression. Together, our results expose a considerable heterogeneity of the CML stem cell population and propose a Lin(-) CD34(+) CD38(-/low) CD45RA(-) cKIT(-) CD26(+) population as a potential therapeutic target for improved therapy response.
|
|
| 19. |
|
|
