HSPVdb—the Human Short Peptide Variation Database for improved mass spectrometry-based detection of polymorphic HLA-ligands

T cell epitopes derived from polymorphic proteins or from proteins encoded by alternative reading frames (ARFs) play an important role in (tumor) immunology. Identification of these peptides is successfully performed with mass spectrometry. In a mass spectrometry-based approach, the recorded tandem mass spectra are matched against hypothetical spectra generated from known protein sequence databases. Commonly used protein databases contain a minimal level of redundancy, and thus, are not suitable data sources for searching polymorphic T cell epitopes, either in normal or ARFs. At the same time, however, these databases contain much non-polymorphic sequence information, thereby complicating the matching of recorded and theoretical spectra, and increasing the potential for finding false positives. Therefore, we created a database with peptides from ARFs and peptide variation arising from single nucleotide polymorphisms (SNPs). It is based on the human mRNA sequences from the well-annotated reference sequence (RefSeq) database and associated variation information derived from the Single Nucleotide Polymorphism Database (dbSNP). In this process, we removed all non-polymorphic information. Investigation of the frequency of SNPs in the dbSNP revealed that many SNPs are non-polymorphic “SNPs”. Therefore, we removed those from our dedicated database, and this resulted in a comprehensive high quality database, which we coined the Human Short Peptide Variation Database (HSPVdb). The value of our HSPVdb is shown by identification of the majority of published polymorphic SNP- and/or ARF-derived epitopes from a mass spectrometry-based proteomics workflow, and by a large variety of polymorphic peptides identified as potential T cell epitopes in the HLA-ligandome presented by the Epstein–Barr virus cells

Concurrent Detection of Circulating Minor Histocompatibility Antigen-Specific CD8+ T Cells in SCT Recipients by Combinatorial Encoding MHC Multimers

Allogeneic stem cell transplantation (SCT) is a potentially curative treatment for patients with hematologic malignancies. Its therapeutic effect is largely dependent on recognition of minor histocompatibility antigens (MiHA) by donor-derived CD8+ T cells. Therefore, monitoring of multiple MiHA-specific CD8+ T cell responses may prove to be valuable for evaluating the efficacy of allogeneic SCT. In this study, we investigated the use of the combinatorial encoding MHC multimer technique to simultaneously detect MiHA-specific CD8+ T cells in peripheral blood of SCT recipients. Feasibility of this approach was demonstrated by applying dual-color encoding MHC multimers for a set of 10 known MiHA. Interestingly, single staining using a fluorochrome- and Qdot-based five-color combination showed comparable results to dual-color staining for most MiHA-specific CD8+ T cell responses. In addition, we determined the potential value of combinatorial encoding MHC multimers in MiHA identification. Therefore, a set of 75 candidate MiHA peptides was predicted from polymorphic genes with a hematopoietic expression profile and further selected for high and intermediate binding affinity for HLA-A2. Screening of a large cohort of SCT recipients resulted in the detection of dual-color encoded CD8+ T cells following MHC multimer-based T cell enrichment and short ex vivo expansion. Interestingly, candidate MiHA-specific CD8+ T cell responses for LAG3 and TLR10 derived polymorphic peptides could be confirmed by genotyping of the respective SNPs. These findings demonstrate the potency of the combinatorial MHC multimer approach in the monitoring of CD8+ T cell responses to known and potential MiHA in limited amounts of peripheral blood from allogeneic SCT recipients

The Human Minor Histocompatibility Antigen1 Is a RhoGAP

The human minor Histocompatibility Antigen HMHA-1 is a major target of immune responses after allogeneic stem cell transplantation applied for the treatment of leukemia and solid tumors. The restriction of its expression to hematopoietic cells and many solid tumors raised questions regarding its cellular functions. Sequence analysis of the HMHA-1 encoding HMHA1 protein revealed the presence of a possible C-terminal RhoGTPase Activating Protein (GAP) domain and an N-terminal BAR domain. Rho-family GTPases, including Rac1, Cdc42, and RhoA are key regulators of the actin cytoskeleton and control cell spreading and migration. RhoGTPase activity is under tight control as aberrant signaling can lead to pathology, including inflammation and cancer. Whereas Guanine nucleotide Exchange Factors (GEFs) mediate the exchange of GDP for GTP resulting in RhoGTPase activation, GAPs catalyze the low intrinsic GTPase activity of active RhoGTPases, resulting in inactivation. Here we identify the HMHA1 protein as a novel RhoGAP. We show that HMHA1 constructs, lacking the N-terminal region, negatively regulate the actin cytoskeleton as well as cell spreading. Furthermore, we show that HMHA1 regulates RhoGTPase activity in vitro and in vivo. Finally, we demonstrate that the HMHA1 N-terminal BAR domain is auto-inhibitory as HMHA1 mutants lacking this region, but not full-length HMHA1, showed GAP activity towards RhoGTPases. In conclusion, this study shows that HMHA1 acts as a RhoGAP to regulate GTPase activity, cytoskeletal remodeling and cell spreading, which are crucial functions in normal hematopoietic and cancer cells

A novel approach to identify antigens recognized by CD4 T cells using complement-opsonized bacteria expressing a cDNA library

In patients with hematological malignancies receiving HLA-matched stem cell transplantation, T cells specific for minor histocompatibility antigens play a major role in graft rejection, induction of graft-versus-host disease and beneficial graft-versus-leukemia reactivity. Several human minor histocompatibility antigens recognized by T cells have been identified, but only two are presented by HLA class II molecules. In search of an efficient approach to identify antigenic peptides processed through the HLA class II pathway, we constructed a cDNA library in bacteria that were induced to express proteins. Bacteria were opsonized with complement to enforce receptor-mediated uptake by Epstein - Barr virus immortalized B cells that were subsequently used as antigen-presenting cells. This approach was validated with an HLA class II-restricted antigen encoded by gene DBY. We were able to identify bacteria expressing DBY diluted into a 300-fold excess of bacteria expressing a nonrelevant gene. Screening of a bacterial library using a DBY-specific CD4 T cell clone resulted in the isolation of several DBY cDNAs. We propose this strategy for a rapid identification of HLA class II-restricted antigenic peptides recognized by CD4 T cells