HLA-DM mediates permissiveness of T-cell alloreactivity to HLA-DPB1
M. Metzing (Essen, DE)
Unrelated donor (UD)-recipient HLA-DPB1 matching status is relevant in UD-hematopoietic stem cell transplantation (UD-HSCT). DPB1 alleles fall into 3 T-cell-epitope-groups (TCE) from high (TCE1) to low (TCE3) immunogenicity which determine permissive (within the same TCE) or non-permissive (across different TCE) mismatches. Permissive HLA-DPB1 TCE mismatches are associated with improved survival post-UD-HSCT and milder in-vitro T-cell alloresponse compared to non-permissive mismatches, however the molecular basis of permissiveness is unknown. The non-classical HLA-molecule HLA-DM is a chaperone altering peptide presentation by classical HLA class II molecules. HLA-DM catalyzes dissociation of low affinity peptides and facilitates binding of high affinity peptides. Here we investigate the effect of HLA-DM on (non-)permissive HLA-DPB1 mismatches by FACS-quantification of CD4+CD137+ T-cell alloresponses from 14 healthy TCE3 homozygous donors to HeLa cells transfected with TCE1 (non-permissive) or TCE3 (permissive) HLA-DPB1 alleles with and without HLA-DM. 84 independent cultures confirmed significantly higher responses to non-permissive vs permissive mismatches in the physiological presence of HLA-DM (p<0.001). Importantly, the pathological absence of HLA-DM significantly raised the magnitude of alloreactivity to permissive mismatches to levels comparable to non-permissive mismatches (p<0.0001), while alloreactivity to non-permissive mismatches was not affected (p=n.s.). FACS-analysis of T-cell receptor (TCR) Vb-families in 36 cultures showed that the median number of highly reacting TCRs present at 60% in the CD4+CD137+ fraction was lower in permissive compared to non-permissive responses in the presence of HLA-DM (3 vs 11), while it was similar in permissive and non-permissive responses in the absence of HLA-DM (14.5 vs 16). Our data suggest that HLA-DM mediates permissiveness of HLA-DPB1 mismatches, an observation with potential clinical implications in UD-HSCT.
Dissecting the relative role of structural and expression polymorphism for T-cell allorecognition of HLA-DPB1
T. Meurer (Essen, DE)
HLA-DPB1 alleles fall into 3 different T cell epitope (TCE) groups, which reflect structural polymorphism in the peptide antigen binding groove. TCE groups determine permissive (in the same TCE) and non-permissive (across different TCE) mismatches relevant in unrelated hematopoietic stem cell transplantation. It has recently been shown that TCE groups are also associated with the rs9277534G/A expression polymorphism in the 3’untranslated region (3’UTR) of HLA-DPB1, suggesting that they might be surrogates of protein expression levels. To test this possibility, we FACS-quantified T-cell alloreactivity from 11 TCE3/3 homozygous healthy individuals to non-permissive TCE1/2 and to permissive TCE3 after 14 day in vitro culture with Hela cells expressing single HLA-DPB1 alleles in the absence of the 3’UTR. Despite similar HLA-DPB1 transcript levels, we observed a significantly higher mean percentage of CD4+ T-cells up-regulating the activation marker CD137 after overnight re-stimulation with non-permissive TCE1/2 compared to permissive TCE3, in 58 independent cultures (38.39% vs 16.54%, p=0.0001). We also separately investigated HLA-DPB1*17:01 (TCE1), *05:01 and *01:01 (both TCE3), in which the common association between TCE groups and rs9277534 expression variant is subverted. When compared to permissive TCE3, significantly higher median levels of T-cell alloreactivity were observed for non-permissive HLA-DPB1*17:01 (16.54% vs 36.13%, p=0.004) but not for permissive HLA-DPB1*05:01 (16.54% vs 26.99%, p=0.119) and *01:01 (16.54% vs 27.8%, p=0.196) in 33 independent cultures, consistent with their classification as TCE1 and TCE3, respectively. These data demonstrate that structural polymorphism of TCE groups is at least in part responsible for their differential immunogenicity independently from expression polymorphism, an observation useful for unrelated donor selection.
HLA-G mediated immune regulation is impaired by a single amino acid exchange at residue 110
A. Celik (Hannover, DE)
The essential role of HLA-G in immune tolerance and its expression frequency in immune privileged tissues is well known; however the specific importance of allelic subtypes in immune responses is still unclear. In total 14 allelic variants exist of which HLA-G*01:01, *01:03 and *01:04 are the most prevalent, differing at residues 31 and 110, respectively. A systematic analysis of these polymorphic residues was the aim of this study. In cytotoxicity assays using artificial K562 cells expressing the HLA-G variants and NK cells as effector cells an increased protective potential of HLA-G*01:04 was observed. The inhibitory receptors ILT-2 and KIR2DL4 are known HLA-G ligands. To analyze binding of HLA-G variants to these molecules, recombinant sHLA-G molecules were used to block inhibitory NK surface receptors. These experiments were repeated reciprocally by blocking or silencing the respective receptor and analyzing the sHLA-G blocking sites. Here the increased protective potential of HLA-G*01:04 could be confirmed, G*01:04 demonstrated a stronger affinity to the inhibitory receptors on NK cells than G*01:01 or G*01:03 which we attribute to their altered peptide binding motifs. HLA-G*01:01, *01:03 and *01:04 restricted peptide profiles were determined utilizing soluble HLA technology and mass spectrometry. All variants exhibit a unique repertoire with marginal overlap, while G*01:04 differs in its peptide anchor profile substantially and appears more invariant than G*01:01 or G*01:03. Using the identified peptides bound to the G*01:04 binding groove, structural analysis explains the impact of the L110I mismatch on the HLA-G conformation and its differential interaction with NK cell receptors. For the first time a contribution of structural alterations within the HLA-G heavy chain for NK cell engagement could be observed. These results will be a step towards understanding immune tolerance and guide towards immune therapeutic strategies.
Visualizing the players of the maternal immune response at the maternal-fetal interface by high-dimensional mass cytometry
A. van der Zwan (Leiden, NL)
In the immunological paradox of pregnancy, the fetus has been considered a semi-allogeneic transplant capable of inducing various immune responses at the maternal-fetal interface. During uncomplicated pregnancies, coordinated interaction between different placental cell types induces immune tolerance, thus preventing a destructive immune response of the mother towards the semi-allogeneic fetus. Most work in the field of reproductive immunology has focused on studying individual placental immune cell subsets, but a system-wide approach that visualizes all major cell lineages simultaneously at different time points during pregnancy is currently lacking. To obtain an unbiased and data-driven overview of the maternal immune cell subsets involved in the local immune responses at the maternal-fetal interface, mass cytometry was applied to perform high-dimensional analysis at the single-cell level. In a pilot study, immune cell heterogeneity within leukocytes isolated from first trimester (6-12 wks) decidual samples was visualized by a t-SNE-based analysis utilizing 36 markers simultaneously, showing that the composition between decidual samples appeared reasonably homogenous. A total of 45 phenotypically distinct subsets were identified consisting of myeloid cells and their progenitors, T cells, a minor subset of B cells and a broad compartment of innate lymphocytes, including innate lymphoid cells. Leukocytes residing in decidual tissue clustered completely separate from leukocytes in peripheral blood, indicating a unique composition of immune cells at the maternal-fetal interface. High-dimensional analysis of the entire immune system that is in play at the maternal-fetal interface, from the beginning of pregnancy until parturition, may have implications for immune-monitoring during pregnancy to prevent possible pregnancy complications such as preterm birth and pre-eclampsia.
Newly identified naturally processed and presented EBV-derived T-cell epitopes are highly immunodominant targets in adoptive immunotherapy for PTLD and EBV-infections
S. Tischer (Hannover, DE)
Morbidity and mortality of immunocompromised patients can be increased by infection with or reactivation of Epstein-Barr-Virus (EBV), which induces development of high-grade EBV+ lymphomas (post-transplant lymphoproliferative disease, PTLD). Adoptive transfer of EBV-specific cytotoxic T cells (EBV-CTLs) has shown promise as a non-toxic immunotherapeutic approach to effectively treat or prevent clinical EBV manifestations. Knowledge of a broad target repertoire is essential to improve the efficacy of immunotherapy and monitoring in high risk patients. This study was designed to identify and evaluate naturally presented A*03:01-restricted EBV-specific T-cell epitopes as effective immunodominant target structures. From EBV-immortalized soluble A*03:01-transduced B cells >1500 peptides were isolated and eleven candidates were pre-selected by different prediction and presentation software tools. T2 and Flex-T peptide-binding and dissociation assays confirmed the stability of peptide/MHC complexes. The immunogenic potential of the candidate peptides and their clinical relevance was evaluated by assessment of frequencies and functionality of EBV-CTLs in healthy donors (n>10) and patients suffering from EBV-associated PTLD (n>5) by multimer staining, IFN-gamma and granzyme B ELI- and FLUROSpot assays. All eleven peptides elicited high EBV-CTL responses in 68.4% of donors. The eligibility for clinical application was determined by small-scale T-cell enrichment using a cytokine secretion assay and phenotypic cell characterization. Mixtures of identified peptides were added to the clinically used EBV consensus pool and showed 5-fold enhancement in stimulation and enrichment efficacy. Thus these eleven newly identified A*03:01-restricted EBV-derived epitopes enlarge the repertoire of known targets and will enable both the manufacturing of clinically applicable EBV-CTLs as well as the monitoring of EBV-specific T-cell responses in patients at risk for viral complications or subsequent to an adoptive transfer.
HLA and KIR mediate susceptibility to Parkinson’s disease
J. Hollenbach (San Francisco, US)
Parkinson’s disease (PD) is a chronic, progressive neurodegenerative disease characterized by the classical motor signs of parkinsonism and a constellation of associated features including autonomic, sensory, cognitive and psychiatric changes. Both underlying immunoregulatory dysfunction and inflammatory processes have been proposed for PD. Under the auspices of the INDIGO (Immunogenetics of Neurological DIseases working GrOup) consortium, we analyzed HLA and KIR variation at high resolution via next-generation sequencing (NGS) methods in 1941 PD cases and 1612 controls. We find that the HLA class II haplotype DRB1*01:01~DQA1*01:01~DQB1*05:01 is predisposing to PD (p<0.005; OR=1.32). While the HLA class II region has been implicated in PD via GWAS and HLA imputation, this is the first large-scale PD study to directly examine HLA sequence-level variation. When KIR3DL1 alleles are examined in combination with HLA in a subset of individuals (1149 cases, 892 controls), we find a strong protective effect (p<0.005; OR=0.20) for individuals homozygous for KIR3DL1*004 in the presence of the Bw4 epitope with Isoleucine at position 80 (Bw4i). Low/null expression alleles defined as a group in combination with Bw4i are also protective in PD (p<0.01; OR=0.73) and protective with respect to age at onset (p=0.001). Our findings point to a role for KIR3DL1 expression level variation, in the presence of the strong HLA ligand Bw4i, with susceptibility to PD. While KIR3DL1*004 may be mis-folded and retained intracellularly, with very low levels of surface expression, in the presence of Bw4i it has been previously demonstrated to be the KIR allele offering the most protection from AIDS progression following infection with HIV. That PD protection is further mediated by low expression alleles implies a common mechanism related to expression of the inhibitory receptor and interaction with the ligand, perhaps due to NK cell licensing. In summary, we find a clear role for HLA class II in predisposition to PD, while protection is afforded by KIR in combination with HLA class I. Taken together, these results strongly support a role for immunogenetic variation in the immunopathology of PD.
The HLA–A and –B landscape of Africa: signatures of pathogen-driven selection and molecular identification of candidate alleles to malaria protection through NGS sequencing
A. Sanchez-Mazas (Geneva 4, CH)
Besides their relevance in clinical transplantation, HLA population genetic studies are essential to understand the molecular evolution of HLA genes and their role in the immune protection or susceptibility of individuals in populations living in heterogeneous environments. The African continent encompasses many diverse climates which condition human lifestyles and expose people to different diseases, among which malaria reaches a disproportionately high prevalence in sub-Saharan countries. Based on case-control studies, several HLA alleles, among which HLA-B*53, were previously found to confer malaria protection but these hypotheses have not been tested within a population genetics framework so far. In order to assess whether P. falciparum malaria could have driven the evolution of HLA frequencies in Africa, we typed the HLA-A and -B loci in approximately 500 individuals from 11 populations of the Sahel belt and we analysed these new data together with those published for 29 other African populations through linear modelling including various geographic and environmental parameters. Our results indicate that, besides extreme genetic differentiation of (semi-)nomadic groups living across the Sahara, likely due to genetic drift, P. falciparum prevalence is a significant predictor of HLA-B*53, B*78 and A*74 frequencies. By using next generation sequencing, we also show that except in one case where B*53:19 is observed, all B*53-positive individuals (N=55), among which 3 homozygotes, are B*53:01:01. As many as 35 of them (64%) also carry C*04:01:01:01, strongly suggesting the presence of a predominant haplotype B*53:01:01~C*04:01:01:01, observed at the homozygous state in one individual from Burkina Faso. B*53:01:01 also exhibits putative associations with C*16:01:01, C*06:02:01:01, A*23:01:01 and A*02:01:01. The other two candidate alleles to malaria protection are predominantly B*78:01 and A*74:01. These results substantially improve our understanding of HLA variation in Africa.
Clinically approved monoclonal antibodies and PM21 particle-stimulated ex vivo-expanded alloreactive natural killer cells: a potent combination against cancer cells
N. Mahaweni (Maastricht, NL)
Natural killer (NK) cell immunotherapy is a promising alternative way to treat cancers. Previously we have shown that a combination of alloreactive (KIR-ligand mismatched) NK cells and Daratumumab - an antibody triggering antibody dependent cellular cytotxicity (ADCC)- could potentiate NK cell reactivity against malignant plasma cells, also in a suppressive tumor microenvironment. Nonetheless, a very large number of activated NK cells will be required to get a desirable clinical response. PM21 particles, derived from the membrane of K562 cells expressing 41BB ligand and membrane-bound interleukin-21 (PM21), accelerate NK cell proliferation ex vivo. In the current study, we investigate the potential combination of PM21 particle-stimulated ex vivo-expanded NK cells and ADCC triggering antibodies. NK cells were expanded from CD3-depleted PBMCs from healthy donors and cultured with or without PM21 particles or a cytokine mix (IL12/IL15/IL18) in the presence of 100 U/ml IL-2 for 3 weeks. NK cells stimulated with PM21 particles expanded 400-600-fold more than NK cells without PM21 particles. Using flow cytometry-based analysis of NK cell phenotypes and cytotoxic potential, we demonstrated that these expanded NK cells express important receptors involved in the anti-tumor response such as KIRs, NKG2A, NKG2D, NKG2C, NKp46, and CD16 (FcgRIII receptor) and efficiently kill cancer target cells. Importantly, addition of clinically approved antibodies further enhanced (20-40%) NK cell reactivity against breast cancer cells (Trastuzumab) and malignant plasma cells (Daratumumab). These observations signify the capability of these PM21-stimulated NK cells to mediate antibody-dependent cell-mediated cytotoxicity. In conclusion, the combination of PM21 particle-stimulated ex vivo-expanded alloreactive natural killer cells and clinically approved antibodies can be an advantageous strategy to refine NK cell immunotherapy against cancers.
DGI Next Generation Award