ABSTRACT. A central dogma over the last 50+ years has been that peptide-binding to HLA-molecules is mediated by the docking of side chains of particular amino acids in the peptide into pockets in the HLA-molecules in a conserved N- to C-terminal orientation. We have shown in our large scale identification of HLA-DP peptides that 9 out of 14 HLA-DP frequent allotypes can present peptides in two orientations that are both functional and can be recognized by CD4 T-Cells. The occurrence of the reverse peptide binding motif, in an N- to C-terminal orientation, is mainly dependent on the DPB1 chains in the allotypes with the DPB1-84DEAV87 sequence at P1. We investigated the role for DPA1 and DPB1 chains by making several cis and trans HLA-DP heterodimers and introducing specific mutations. When we analyzed the presentation of peptides by peptides elution we found that not only the sequence of the DPB1 chain is crucial in the presentation of peptides in the reversed orientation, but we also showed a key role for the DPA1 chain in modifying the peptidome. This leads to the conclusion that specific heterozygous HLA-DP peptidomes will be shaped depending on the potential cis and trans heterodimer-combinations. In the context of HLA-DP-mismatched allogeneic stem cell transplantation, mismatched HLA-DP alleles can provoke profound allo-HLA-DP-specific immune responses from the donor T-cell repertoire dependent of the composition of the mismatched peptidome. Our results illustrate unique features of HLA-DP molecules that substantially broaden the HLA-class II bound peptide repertoire to combat pathogens, eliminate cancer cells and can have significant influence on the outcome of allogeneic stem cell transplantation.

ABSTRACT. Allogeneic Hematopoietic Cell Transplantation (allo-HCT) represents the most successful therapeutic option for many patients suffering from Acute Myeloid Leukemia (AML). Nevertheless, tumor cells frequently enact strategies to evade immune recognition and re-emerge, by either by impairing their recognition mediated by T cells through reduced antigen presentation and enforcement of inhibitory checkpoints, or by taking advantage of their own tumor microenvironment (TME). Here, we used single-cell RNA-sequencing to detail the changes that occur in the bone marrow of patients who experience AML relapse after allo-HCT. We profiled 114,273 cells from 18 AML patients with different mechanisms of leukemia relapse, including downregulation of HLA class II (n=5), upregulation of inhibitory ligands (n=4), and HLA loss (n=9), 10,080 cells from 3 healthy donors and 16,832 cells from 3 post-transplant AML patients in complete remission. First, we used different gene signatures representing successive stages of normal hematopoietic development to calculate a gene module score for each relapse modality. HLA loss relapses showed a more immature expression profile, suggesting that the hematopoietic cell of origin impacts on the mechanism of relapse. Focusing on the TME, we analyzed monocytes, and observed the presence of an interesting cluster of donor origin characterized by an anti-inflammatory gene signature, which might play a facilitating role in leukemia immune evasion. Lastly, we leveraged on TCR sequencing to describe the complexity of the T cell repertoire in different relapse settings, and found out that AML relapses characterized by HLA class II downregulation displayed a more clonal TCR repertoire. Findings from this study will improve our understanding of how leukemic cells exploit their TME to escape immune surveillance in the different patients, and identify new vulnerabilities to be exploited for personalized therapeutic approaches.

ABSTRACT. Epstein-Barr virus (EBV) infects more than 90% of the population and remains in B-cell compartments life-long, passing through several latency stages. While in healthy individuals strong immune responses control EBV reactivation, in immunocompromised patients, infections and reactivations can lead to severe EBV-associated malignant complications, such as post-transplant lymphoproliferative disease (PTLD). These patients may benefit from EBV-specific T-cell therapy. In latency stages II/III, latent membrane protein 2A (LMP2A) is expressed and therefore associated with PTLD as well as various lymphomas and carcinomas. Recently a clinically protective TCR recognizing an LMP2A-derived peptide in context of HLA-A*02 was identified. Based on this, we developed TCR-engineered T cells and further equipped these with an inducible cassette for locally restricted IL-18 release (LMP2A_iIL18_TCR-Ts), which was shown to convert T cells into pro-inflammatory effector cells, preventing loss of function and exhaustion, and redirecting the immunosuppressive tumor microenvironment. As LMP2A_iIL18_TCR-T functionality is hypothesized to be superior compared to endogenous T cells, we analyzed their phenotype, replicative capacity, activation and exhaustion state as well as cytotoxicity towards HLA-A*02+EBV+ target cells. In this context, LMP2A_iIL18_TCR-Ts showed HLA-A*02:LMP2A specificity without signs of HLA cross-reactivity or recognition of an irrelevant HLA-A*02-restricted peptide, which was further confirmed by Ca2+ flux analysis. Combination with inducible IL-18 expression increased cytotoxicity measured by 7-AAD staining, live cell imaging and detachment of target cells in real-time impedance measurements. In conclusion, ex vivo isolated protective TCRs could be redirected into T cells from third-party donors with the potential to attract innate immune cells and alter the tumor environment, thereby widening the applicability of T-cell therapy to refractory viral infections.

ABSTRACT. Measure of drug-mediated immune reactions that are dependent on the patient’s genotype determine individual medication protocols. Despite extensive clinical trials prior to the license of a drug, certain immune reactions cannot be predicted. The need for acknowledgement of the proteomic state for selected individuals under drug administration becomes obvious. The association between certain HLA molecules and drugs or their metabolites has been analyzed in recent years. Dependent on the patient’s genotype, Carbamazepine (CBZ) hypersensitivities cause diverse disease pictures as MPE, DRESS or more severe diseases SJS or TEN. Not only the association between HLA-B*15:02 or HLA-A*31:01 but also between HLA-B*57:01 and CBZ administration could be demonstrated. This study illuminates the mechanism of HLA-B*57:01-mediated CBZ hypersensitivity by full proteome analysis. Genetically engineered human B-LCLs expressing sHLA-B*57:01 molecules were treated with CBZ or its metabolite carbamazepine-10,11-epoxide (EPX). The incapability of these B-LCLs to metabolize CBZ makes them an ideal system for analyzing the influence of CBZ and EPX orthogonally. HLA/drug complexes were purified and the availability of CBZ or EPX was monitored by mass spectrometry. The data reveal significant stronger engagement of B*57:01 to EPX than to CBZ. Subsequent full proteome analysis of engineered B*57:01+ cells following CBZ or EPX treatment uncovered an unknown mechanisms; EPX introduced drastic proteomic alterations as the induction of inflammatory processes through the upstream kinase ERBB2 and the upregulation of NFκB and JAK/STAT pathway implying a pro-apoptotic, pro-necrotic shift in the cellular response. Simultaneously, anti-inflammatory pathways and associated effector proteins were downregulated. Fundamental knowledge of drug-susceptible HLA molecules, their drug antagonist and the disequilibrium of the proteomic content will certainly facilitate personalized and safe medication.

ABSTRACT. Downregulation of Major Histocompatibility Complex (MHC) molecules is often key to evasion of the immune system by tumours and viruses. Transmissible cancers transmit between individuals, in a manner akin to a metastatic event, providing a unique opportunity to study the evolution of MHC loss in the face of selective pressure from the immune system. Tasmanian devils are infected with two genetically distinct transmissible cancers which transmit via biting behaviours. Devil Facial Tumour Disease (DFT1) emerged over 25 years ago, while DFT2 was identified in 2014. In contrast to DFT1, DFT2 tumours express MHC class I molecules; but recent evidence shows some are MHC class I negative, suggestive of evolving immune escape by this cancer. We hypothesise that as DFT2 spreads through the population, encountering hosts with disparate MHC class I genotypes, selection for MHC class I loss is occurring. Here we have used immunohistochemistry to assess MHC class I expression and CD3+ cell infiltration in DFT2 primary tumour biopsies (n=26). Classical MHC class I expression was highly variable between tumours, with 9 tumours negative by immunohistochemistry. Tumours were also variable for expression of 2 non-classical MHC class I genes, which could play a role in immunosuppression. To investigate drivers of MHC loss we have genotyped (n=17) host animals at three classical MHC class I loci. Host-tumour comparisons at MHC class I reveal potential immunogenic alleles driving immune escape in DFT2. These results demonstrate that DFT2 is evolving immune evasion mechanisms as it transmits between individuals in the population, with the potential for more rapid dispersion if MHC-negative subclones gain dominance. However, this data can be used to predict DFT2 transmission dynamics and inform more effective management of the population. Further, this study provides a platform to investigate the mechanisms behind MHC loss in a cancer under sustained pressure from the immune system.

ABSTRACT. The HLA region is the hallmark of balancing selection, argued to be driven by the pressure to present a wide variety of viral epitopes. As such the peptide-binding positions have been the center of interest for the detection of selection. However, the human MHC molecule also directly binds to the T-Cell Receptor and KIR. We here use the HLA allele frequencies in over six-million donors with a novel machine learning based method to estimate selection to show that: A) the allele frequency can be predicted from its sequence; B) The strongest selection is actually in KIR binding regions, followed by the peptide-binding cleft; C) the selection from the direct interaction with the KIR or TCR is centered on positively charged residues (mainly Arginine); and D) some positions in the peptide-binding cleft are not associated with the allele frequency, especially the ones with Tyrosine residues. These results suggest that the balancing selection for peptide presentation may be combined with a positive selection for KIR and TCR binding.

ABSTRACT. In solid organ transplantation, the formation of de novo donor-specific antibodies (dnDSA) is associated with inferior graft function and survival. dnDSA are induced by mismatched configurations of polymorphic amino acids, often referred to as eplets. While eplet mismatches have been associated with dnDSA formation, not every eplet mismatch appears to be immunogenic. To implement HLA eplet matching in allocation in order to prevent dnDSA formation, it is essential to define non- and immunogenic eplet mismatches. Therefore, the immunogenic epitope project of the 18th IHIWS was aimed to define the most immunogenic HLA eplet mismatches. Data from first-time, non-sensitised solid organ transplant recipients that either developed dnDSA or not after transplantation were collected. High resolution HLA typing of recipient and donor, and luminex single antigen bead results pre- and post-transplantation, at first detection of dnDSA, or follow up of ≥5 years without DSA were collected. For each mismatched donor HLA allele, eplet mismatches were determined using HLA-EMMAv2.0beta, which contains all eplets from the HLA Eplet Registry. Subsequently, we determined how often a specific eplet was mismatched and how often it resulted in dnDSA (DSA count). A total number of 809 patient donor couples was submitted to the IHIWS component. In these couples, certain eplets were never mismatched and considerable overlap in the number of eplet mismatches between the no DSA and dnDSA group was observed. Many eplet mismatches never resulted in antibody formation. Per locus the immunogenicity score for each eplet per locus was defined by dividing DSA count by the eplet mismatch count from which the most immunogenic eplets could be defined. While a first inventory of the most immunogenic eplet mismatches was made, extending 18th IHIW cohort with recipients from other HLA backgrounds during the 19th IHIWS is crucial to move towards implementing eplet matching in clinical transplantation.

ABSTRACT. Introduction: Oocyte donation (OD) pregnancies are related to a higher degree of fetal-maternal HLA mismatching and a higher risk of complications compared to naturally conceived pregnancies. Nevertheless, many OD pregnancies maintain healthy until term. We hypothesize that in OD pregnancies with high HLA dissimilarity, the immune response at the fetal-maternal interface (the decidua) is divergent to maintain a healthy state. Here we focus on myeloid cells as we previously found these cells to be highly frequent in the decidua and they are essential in maintaining a healthy pregnancy. Methods: We performed imaging mass cytometry (IMC) using a 42-antibody panel on decidua tissues of 8 uncomplicated singleton OD pregnancies. Single cell masks were created using cell segmentation. Child and mother were typed for HLA-A, -B, C, -DRB1, and - DQB1, and fetal-maternal HLA mismatches were calculated. Based on the number of HLA mismatches, samples were separated into a semi-allogeneic group (≤5 HLA mismatches, n=4) and a fully allogeneic group (>5 mismatches, n=4). Results: Myeloid cells represented the most abundant (~60%) immune cell population in the decidua. Thirteen phenotypically distinct subclusters were identified within the myeloid cell lineage. The IMC gave a possibility to study the microenvironment of each cell. The fully allogeneic group showed a higher frequency of maternal myeloid cells in the maternal T cells microenvironment than the semi-allogeneic OD group (p<0.050). Most notably, a higher extent of interaction between CD163+CD206+HLA-DR- myeloid cells and CD4+ T cells was observed in the fully allogeneic group (p<0.050). Conclusions: Our results show the phenotypic diversity of decidual myeloid cells and their prominent frequency in uncomplicated OD pregnancies. By interacting with T cells, decidual myeloid cells might perform immune regulatory functions to compensate for the higher fetal-maternal HLA mismatch load in OD pregnancies.