Diana Paola Granados

ER stress affects processing of MHC class I-associated peptides

BackgroundViral infection and neoplastic transformation trigger endoplasmic reticulum (ER) stress. Thus, a large proportion of the cells that must be recognized by the immune system are stressed cells. Cells respond to ER stress by launching the unfolded protein response (UPR). The UPR regulates the two key processes that control major histocompatibility complex class I (MHC I)-peptide presentation: protein synthesis and degradation. We therefore asked whether and how the UPR impinges on MHC I-peptide presentation.ResultsWe evaluated the impact of the UPR on global MHC I expression and on presentation of the H2Kb-associated SIINFEKL peptide. EL4 cells stably transfected with vectors coding hen egg lysozyme (HEL)-SIINFEKL protein variants were stressed with palmitate or exposed to glucose deprivation. UPR decreased surface expression of MHC I but did not affect MHC I mRNA level nor the total amount of intracellular MHC I proteins. Impaired MHC I-peptide presentation was due mainly to reduced supply of peptides owing to an inhibition of overall protein synthesis. Consequently, generation of H2Kb-SIINFEKL complexes was curtailed during ER stress, illustrating how generation of MHC I peptide ligands is tightly coupled to ongoing protein synthesis. Notably, the UPR-induced decline of MHC I-peptide presentation was more severe when the protein source of peptides was localized in the cytosol than in the ER. This difference was not due to changes in the translation rates of the precursor proteins but to increased stability of the cytosolic protein during ER stress.ConclusionOur results demonstrate that ER stress impairs MHC I-peptide presentation, and that it differentially regulates expression of ER- vs. cytosol-derived peptides. Furthermore, this work illustrates how ER stress, a typical feature of infected and malignant cells, can impinge on cues for adaptive immune recognition.

Deletion of Immunoproteasome Subunits Imprints on the Transcriptome and Has a Broad Impact on Peptides Presented by Major Histocompatibility Complex I molecules

Proteasome-mediated proteolysis plays a crucial role in many basic cellular processes. In addition to constitutive proteasomes (CPs), which are found in all eukaryotes, jawed vertebrates also express immunoproteasomes (IPs). Evidence suggests that the key role of IPs may hinge on their impact on the repertoire of peptides associated to major histocompatibility complex (MHC) I molecules. Using a label-free quantitative proteomics approach, we identified 417 peptides presented by MHC I molecules on primary mouse dendritic cells (DCs). By comparing MHC I-associated peptides (MIPs) eluted from primary DCs and thymocytes, we found that the MIP repertoire concealed a cell type-specific signature correlating with cell function. Notably, mass spectrometry analyses of DCs expressing or not IP subunits MECL1 and LMP7 showed that IPs substantially increase the abundance and diversity of MIPs. Bioinformatic analyses provided evidence that proteasomes harboring LMP7 and MECL1 have specific cleavage preferences and recognize unstructured protein regions. Moreover, while differences in MIP repertoire cannot be attributed to potential effects of IPs on gene transcription, IP subunits deficiency altered mRNA levels of a set of genes controlling DC function. Regulated genes segregated in clusters that were enriched in chromosomes 4 and 8. Our peptidomic studies performed on untransfected primary cells provide a detailed account of the MHC I-associated immune self. This work uncovers the dramatic impact of IP subunits MECL1 and LMP7 on the MIP repertoire and their non-redundant influence on expression of immune-related genes.

MHC I–associated peptides preferentially derive from transcripts bearing miRNA response elements

MHC I-associated peptides (MIPs) play an essential role in normal homeostasis and diverse pathologic conditions. MIPs derive mainly from defective ribosomal products (DRiPs), a subset of nascent proteins that fail to achieve a proper conformation and the physical nature of which remains elusive. In the present study, we used high-throughput proteomic and transcriptomic methods to unravel the structure and biogenesis of MIPs presented by HLA-A and HLA-B molecules on human EBV-infected B lymphocytes from 4 patients. We found that although HLA-different subjects present distinctive MIPs derived from different proteins, these MIPs originate from proteins that are functionally interconnected and implicated in similar biologic pathways. Secondly, the MIP repertoire of human B cells showed no bias toward conserved versus polymorphic genomic sequences, were derived preferentially from abundant transcripts, and conveyed to the cell surface a cell-type-specific signature. Finally, we discovered that MIPs derive preferentially from transcripts bearing miRNA response elements. Furthermore, whereas MIPs of HLA-disparate subjects are coded by different sets of transcripts, these transcripts are regulated by mostly similar miRNAs. Our data support an emerging model in which the generation of MIPs by a transcript depends on its abundance and DRiP rate, which is regulated to a large extent by miRNAs.

Impact of genomic polymorphisms on the repertoire of human MHC class I-associated peptides

For decades, the global impact of genomic polymorphisms on the repertoire of peptides presented by major histocompatibility complex (MHC) has remained a matter of speculation. Here we present a novel approach that enables high-throughput discovery of polymorphic MHC class I-associated peptides (MIPs), which play a major role in allorecognition. On the basis of comprehensive analyses of the genomic landscape of MIPs eluted from B lymphoblasts of two MHC-identical siblings, we show that 0.5% of non-synonymous single nucleotide variations are represented in the MIP repertoire. The 34 polymorphic MIPs found in our subjects are encoded by bi-allelic loci with dominant and recessive alleles. Our analyses show that, at the population level, 12% of the MIP-coding exome is polymorphic. Our method provides fundamental insights into the relationship between the genomic self and the immune self and accelerates the discovery of polymorphic MIPs (also known as minor histocompatibility antigens).

Origin and plasticity of MHC I-associated self peptides.

Endogenous peptides presented by MHC I molecules represent the essence of self for CD8 T lymphocytes. These MHC I peptides (MIPs) regulate all key events that occur during the lifetime of CD8 T cells. CD8 T cells are selected on self-MIPs, sustained by self-MIPs, and activated in the presence of self-MIPs. Recently, large-scale mass spectrometry studies have revealed that the self-MIP repertoire is more complex and plastic than previously anticipated. The composition of the self-MIP repertoire varies from one cell type to another and can be perturbed by cell-intrinsic and -extrinsic factors including dysregulation of cellular metabolism and infection. The complexity and plasticity of the self-MIP repertoire represent a major challenge for the maintenance of self tolerance and can have pervasive effects on the global functioning of the immune system.

MHC class I–associated peptides derive from selective regions of the human genome

MHC class I-associated peptides (MAPs) define the immune self for CD8+ T lymphocytes and are key targets of cancer immunosurveillance. Here, the goals of our work were to determine whether the entire set of protein-coding genes could generate MAPs and whether specific features influence the ability of discrete genes to generate MAPs. Using proteogenomics, we have identified 25,270 MAPs isolated from the B lymphocytes of 18 individuals who collectively expressed 27 high-frequency HLA-A,B allotypes. The entire MAP repertoire presented by these 27 allotypes covered only 10% of the exomic sequences expressed in B lymphocytes. Indeed, 41% of expressed protein-coding genes generated no MAPs, while 59% of genes generated up to 64 MAPs, often derived from adjacent regions and presented by different allotypes. We next identified several features of transcripts and proteins associated with efficient MAP production. From these data, we built a logistic regression model that predicts with good accuracy whether a gene generates MAPs. Our results show preferential selection of MAPs from a limited repertoire of proteins with distinctive features. The notion that the MHC class I immunopeptidome presents only a small fraction of the protein-coding genome for monitoring by the immune system has profound implications in autoimmunity and cancer immunology.

Proteogenomic-based discovery of minor histocompatibility antigens with suitable features for immunotherapy of hematologic cancers.

Pre-clinical studies have shown that injection of allogeneic T cells primed against a single minor histocompatibility antigen (MiHA) could cure hematologic cancers (HC) without causing any toxicity to the host. However, translation of this approach in humans has been hampered by the paucity of molecularly defined human MiHAs. Using a novel proteogenomic approach, we have analyzed cells from 13 volunteers and discovered a vast repertoire of MiHAs presented by the most common HLA haplotype in European Americans: HLA-A*02:01;B*44:03. Notably, out of >6000 MiHAs, we have identified a set of 39 MiHAs that share optimal features for immunotherapy of HCs. These ‘optimal MiHAs’ are coded by common alleles of genes that are preferentially expressed in hematopoietic cells. Bioinformatic modeling based on MiHA allelic frequencies showed that the 39 optimal MiHAs would enable MiHA-targeted immunotherapy of practically all HLA-A*02:01;B*44:03 patients. Further extension of this strategy to a few additional HLA haplotypes would allow treatment of almost all patients.