Project description:A high-throughput mass spectrometry analysis was used to identify more than 16,000 cell peptides bound to several HLA-DR and -DP class II molecules isolated from large amounts of two human cell lines (HOM-2 and JY).
Project description:The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) poses a major challenge in hemophilia A (HA) treatment. The formation of FVIII inhibitors is a CD4+ T-cell-dependent mechanism which includes anti- gen presenting cells (APC), B- and T-helper lymphocytes. APC present FVIII-derived peptides on major histocompatibility complex class II (MHC-II) to CD4+ T cells. We previously established a mass spectrometry-based approach to delineate the FVIII repertoire presented on HLA-DR and HLA-DQ. In this study, specific attention was directed towards the identification of FVIII peptides presented on HLA-DP. A data-set of naturally processed FVIII peptides was generated by incubating human FVIII with immature monocyte-derived dendritic cells (moDC) from HLA-typed healthy donors. Using this method, we iden- tified 176 to 1,352 different HLA-DP presented peptides per donor, including 26 different FVIII-derived peptides. The most frequently presented peptides derived from the A3 and C2 domains of FVIII. Comparison of the FVIII repertoire presented on HLA-DP with that presented on HLA-DR revealed considerable overlap but also suggested preferential presentation of specific peptides on either HLA-DR or HLA-DP. Fourteen FVIII peptides presented on HLA-DP were synthesized and evalu- ated for their binding ability to the commonly expressed HLA-DP4 molecule which is highly prevalent in the Caucasian population. Peptide binding studies showed that 7 of 14 peptides competed with a reference peptide to HLA-DP4. Interest- ingly, an A3 domain-derived peptide bound with high affinity to HLA-DP4, positioning this peptide as a prime candidate for the development of novel peptide-based tolerogenic strategies for FVIII inhibitors.
Project description:Accurate prediction of antigen presentation by Human Leukocyte Antigen (HLA) class II molecules is crucial for rational development of immunotherapies and vaccines targeting CD4 T cell activation. So far, most prediction methods for HLA class II antigen presentation have focused on HLA-DR due to limited availability of immunopeptidomics data for HLA-DQ and HLA-DP, while not taking into account alternative peptide binding modes. Here, we present an update to the NetMHCIIpan prediction method which closes the performance gap between all three HLA class II loci. We accomplish this by first integrating large immunopeptidomics datasets describing the HLA class II specificity space across loci using a refined machine learning framework that accommodates inverted peptide binders. Next, we apply targeted immunopeptidomics assays to generate novel data that covers additional HLA-DP specificities. The final method, NetMHCIIpan-4.3, achieves high accuracy and molecular coverage across all HLA class II allotypes.
Project description:HLA-DR-lacking HSPCs [HLA-DR(-) HSPCs] were detected in aplastic anemia (AA) patients with HLA-DR15. HLA-DR(-) HSPCs may evade the attack by CD4+ T-cells recognizing the autoantigen presented by HLA-DR15. The goal of this study is to clarify the immune escape mechanisms from antigen-specific T-cells by comparing the trranscriptome profile of HLA-DR(+) HSPCs and HLA-DR(-) HSPCs.
Project description:Myeloid-derived suppressor cells (MDSC) is a heterogeneous population of cells that can negatively regulate T-cell function. As opposed to murine MDSC, which are characterized as Gr-1+CD11b+ cells, human MDSC are not so clearly defined due to lack of specific markers. Our lab has previously identified a new subset of MDSC as CD14+HLA-DR-neg/low cells from PBMC. CD14+HLA-DR-neg/low MDSC not only suppress proliferation and IFN-gamma secretion of autologous T cells, but also induce CD25+Foxp3+ regulatory T cells that are suppressive in vitro, whereas the counterpart CD14+HLA-DR-high monocytes don’t have the effect. In this study, we compare the immune-related gene expression between CD14+HLA-DR-neg/low MDSC and CD14+HLA-DR-high monocytes to better characterize the difference between these two populations and to find new potential specific marker for human MDSC. PBMC were isolated from fresh blood healthy donor by density centrifugation. CD14+ cells were isolated by AutoMACS CD14 microbeads using a AutoMACS (Miltenyi), and then stained with CD14 and HLA-DR antibodies. MDSC and monocytes control cells were sorted as CD14+ HLA-DR-neg/low and CD14+HLA-DR-high cells respectively. The sorted two populations were immediately frozen in liquid nitrogen and shipped to the company on dry ice for RNA isolation and further microarray.
Project description:Negative control (healthy cells) of MSV000079647. HLA-DR ligands from healthy Jy cells. Using mass spectrometry analysis of complex HLA class II-bound peptide pools isolated from large amounts of non-infected cells were identified.
Project description:Myeloid-derived suppressor cells (MDSC) is a heterogeneous population of cells that can negatively regulate T-cell function. As opposed to murine MDSC, which are characterized as Gr-1+CD11b+ cells, human MDSC are not so clearly defined due to lack of specific markers. Our lab has previously identified a new subset of MDSC as CD14+HLA-DR-neg/low cells from PBMC. CD14+HLA-DR-neg/low MDSC not only suppress proliferation and IFN-gamma secretion of autologous T cells, but also induce CD25+Foxp3+ regulatory T cells that are suppressive in vitro, whereas the counterpart CD14+HLA-DR-high monocytes don’t have the effect. In this study, we compare the immune-related gene expression between CD14+HLA-DR-neg/low MDSC and CD14+HLA-DR-high monocytes to better characterize the difference between these two populations and to find new potential specific marker for human MDSC.
Project description:HLA-DRB1 alleles have been associated with several autoimmune diseases. In anti-citrullinated protein antibody positive rheumatoid arthritis (ACPA-positive RA), HLA-DRB1 shared epitope (SE) alleles are the major genetic risk factors. In order to investigate whether expression of different alleles of major histocompatibility complex (MHC) Class II genes influence functions of immune cells, we investigated transcriptomic profiles of a variety of immune cells from healthy individuals carrying different HLA-DRB1 alleles. Sequencing libraries from peripheral blood mononuclear cells, CD4+ T cells, CD8+ T cells, and CD14+ monocytes of 32 genetically pre-selected healthy female individuals were generated, sequenced and reads were aligned to the standard reference. For the MHC region, reads were mapped to available MHC reference haplotypes and AltHapAlignR was used to estimate gene expression. Using this method, HLA-DRB and HLA-DQ were found to be differentially expressed in different immune cells of healthy individuals as well as in whole blood samples of RA patients carrying HLA-DRB1 SE-positive versus SE-negative alleles. In contrast, no genes outside the MHC region were differentially expressed between individuals carrying HLA-DRB1 SE-positive and SE-negative alleles. Existing methods for HLA-DR allele-specific protein expression were evaluated but were not mature enough to provide appropriate complementary information at the protein level. Altogether, our findings suggest that immune effects associated with different allelic forms of HLA-DR and HLA-DQ may be associated not only with differences in the structure of these proteins, but also with differences in their expression levels.