Tapasin - The star of the show in HLA-I maturation

Human leukocyte antigen class I (HLA-I) molecules are present on the cell surface of all nucleated cells. They bind intracellular peptides and present them to the immune system thus providing a readout to cytotoxic T-cells about the status of a cell. Maturation of the HLA-I molecule includes peptide binding in the endoplasmic reticulum with the assistance of several other proteins collectively called the antigen processing machinery (APM). One of these proteins, tapasin, is of particular interest as it is dedicated to HLA-I and plays a key role in the selection and binding of peptides that induce stable HLA-I molecules. In this thesis I have studied HLA-I maturation, and tapasin in particular, both biochemically and in tumor material. The first paper is a study of HLA-I folding in the presence and absence of a part of recombinant tapasin (tpn1-87). Here we studied how different HLA-I allomorphs depend on tapasin and the influence of peptide length. This is the first study where a large number of HLA-I allomorphs have been simultaneously analyzed for tapasin facilitation. We found that the influence of peptide length for the different allomorphs increased with their tapasin dependence. In paper II and III we studied HLA-I, tapasin and other APM proteins in tumor sections and cell lines of glioblastoma multiforme (GBM). In tumors APM proteins are commonly downregulated as a strategy to evade the immune system. We also found the APM proteins in our tumor material to be highly dysregulated with strongly linked HLAI and tapasin expression. In tumor tissue sections HLA-I and tapasin expression also correlated with survival of GBM patients. High resolution HLA-I typing allowed us to study the HLA-I allomorphs expressed in GBM patients and also each allomorph’s tapasin dependence. We found that GBM patients display HLA-I allomorph profiles with mixed dependency of tapasin, similar as in a healthy cohort. In addition we show that tapasin deficient cells present suboptimally loaded HLA-I molecules on the cell surface. By exogenous addition of high affinity peptides we were able to increase the stability of presented HLA-A*02:01 molecules. The tapasin dependency of each allomorph as well as composition and proportions of HLA-I allomorphs presented on the cell surface is of importance not only for mechanistic understanding but also for immunotherapy settings in different diseases. GBM is an aggressive brain tumor with poor prognosis and there are high demands for new and more effective treatments. We propose individualized immunotherapy protocols where tapasin expression and tapasin dependency of allomorphs expressed in each patient are taken into consideration to improve the selection of peptide:HLA-I combinations for peptide vaccines

HLA-I Antigen Presentation and Tapasin Influence Immune Responses Against Malignant Brain Tumors - Considerations for Successful Immunotherapy.

Human leukocyte antigen class I (HLA-I) presents antigenic peptides to cytotoxic CD8+ T cells (CTLs). This is a pivotal step in the generation of CTL responses. Both the quantity and quality of peptide-HLA-I (pHLA-I) complexes are crucial for CTL responses, but the level of HLA-I expression per se is also directly involved in dictating NK-cell responses. Antigen processing machinery (APM) proteins are involved in the maturation of HLA-I and in the selection of which peptides are - or are not - presented. Thus, these proteins are key players in shaping the immune response to cells in health and disease. In this review, we recap the most important features of APM components and their synergistic work to assure proper pHLA-I cell surface expression. We pay special attention to the HLA-I dedicated multifunctional protein, tapasin, and in relation to the different tapasin-dependency of HLA-I allomorphs we also discuss allomorph specific traits in maturation, structure and linkage to malignant diseases and brain tumors in particular. We next discuss the possibilities of restoring or manipulating the immune responses against brain tumors. In this context we discuss IFNγ therapy, cytostatics and irradiation. Finally, we integrate current views and knowledge to set the direction for future emphasis in the area of immunotherapy against brain tumors

Tapasin and human leukocyte antigen class I dysregulation correlates with survival in glioblastoma multiforme.

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

Tapasin facilitation of MHC-I separates closely related allomorphs, is strongly influenced by peptide length and depends on stability

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

Tapasin Facilitation of Natural HLA-A and -B Allomorphs Is Strongly Influenced by Peptide Length, Depends on Stability, and Separates Closely Related Allomorphs.

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