56. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

Vorsitz

Prof. Dr.

Soluble components of the ECP product affect monocyte activation in vitro

Background Extracorporeal photopheresis (ECP) is an effective therapy for various T-cell-mediated diseases. However, its mechanism of action remains largely unknown. Most studies focus on fate and function of cellular components of ECP product and prove their contribution to ECP efficacy, whereas the cell free fraction of the ECP product has not been thoroughly studied. This study further investigated the composition of soluble factors of the ECP product and their functional effect on monocytes in vitro.Methods Human peripheral blood mononuclear cells (PBMCs) of healthy blood donors were incubated with 200ng/ml 8-methoxypsoralen (8-MOP) for 30 min and treated with 2 J/cm2 UVA in vitro. PBMCs treated with either 8-MOP, UVA or left untreated served as controls. Up to 48 hours later, cell free cell culture supernatant (ECP-SN) was harvested. Concentrations of several cytokines within the ECP-SN were analysed using a cytometric bead-based immunoassay. To address the functional effect of the soluble ECP product on immune cells, PBMCs were exposed to ECP-SN alone for 12h, 24h and 48h or exposed to ECP-SN for 24h and then stimulated with R848 or INF-γ for 24h. Survival and activation marker expression on monocytes were analysed via flow cytometry.Results ECP treatment of PBMCs affects the cytokine milieu in cell culture supernatant over time compared to controls: Concentration of IL-6 and IL-1β were increased and MCP-1 concentration was decreased in ECP-SN compared to controls within 48h, whereas IL-8 IL-10 and IL-2 were not affected. Regarding the functional analysis of the ECP-SN, addition of ECP-SN to PBMCs induced increases cell death within 48 hours in non-monocytes. Moreover, ECP-SN activates monocytes reflected by increased CD38 and CD83 expression and further increases CD86 expression induced by R848 or INF-γ stimulation.Conclusion These data suggest that soluble factors may play a role in the mechanism of action of ECP in addition to cell-based effects described in the literature. Further studies exploring additional soluble factors within the ECP product, or investigating its functional effects on other cell types can contribute to a better understanding of the immunological effects induced by ECP therapy. Finally, this could help to improve ECP therapy and to expand its medical scope.Offenlegung Interessenkonflikt: Es besteht kein Interessenkonflikt.

Immunotherapy

Freier Vortrag (Reinhold-Eckstein-Investigator-Award)

Survival of dendritic cell subsets is differentially affected by ECP treatment in vitro

Background Extracorporeal photopheresis (ECP) can induce both an activating as well as regulatory immune response depending on the clinical context. Current studies indicate that dendritic cells (DC) – key players in the regulation of T cell immunity – are critical for ECP efficacy. However, contribution of DC subsets to ECP efficacy is scarcely investigated. This study examined the direct effect of ECP treatment on plasmacytoid DC (pDC) and conventional DC type 1 (cDC1) and type 2 (cDC2) in vitro.Methods To address the effect of ECP treatment on DC subsets, human peripheral blood mononuclear cells (PBMC) of healthy blood donors were treated with 200ng/ml 8-Methoxypsoralen (8-MOP) for 30 min and then irradiated with 2J/cm2 UVA in vitro. Untreated, UVA or 8-MOP treated PBMCs served as controls. After 24 and 48 hours quantity and quality of cell death of pDC, cDC1 and cDC2 was evaluated via flow cytometry and compared to the survival of other leukocytes. To reliably identify cDC1 within PBMCs after in vitro culture, staining with XCR1, CLEC9A and CD141 was compared via flow cytometry.Results Staining of PBMC with CD141 was superior to CLEC9A and XCR1 to identify cDC1 after in vitro culture. Thus, after exclusion of CD45+Lin+CD14+CD16+ cells, CD123+HLA+ were defined as pDC, CD123-CD11c+HLA+CD1c+ cells as cDC2 and CD123-CD11c+HLA+CD141+ cells as cDC1. cDC1 and cDC2 were more sensitive to cell death due to in vitro culture compared to pDC and other leukocyte subsets over time. T cells, B cells and pDCs were similar susceptible to ECP treatment. Relative amount and absolute number of these populations was substantially reduced within 48h. In contrast, cDCs were less affected by ECP induced cell death.Conclusion The results of this study indicate that DC subsets respond differentially to ECP treatment. Thus, it seems to be worth to further investigate the role of pDC, cDC1 and cDC2 during ECP treatment in more detail. Such studies will help to get a better understanding of the immunological mechanism of action of ECP and therefore can contribute to improve this therapy.Offenlegung Interessenkonflikt: The authors declare that they have no competing interests.

Freier Vortrag

Vitamin C conditioning creates CAR-T cells with superior cytotoxic capacity and metabolic fitness to combat the immunosuppressive tumor micromilieu

Background Despite the promising results of CAR-T-cell therapy, poor in vivo persistence and low potency in the treatment of solid tumors are challenges that remain to be addressed. It has been shown that the efficacy of adoptively transferred and genetically modified T cells can be modulated by ex vivo culture conditions. Vitamin C (vitC) is a micronutrient that influences the immune system by mechanisms such as the regulation of epigenetic processes and reactive oxygen species-induced oxidative stress.Methods In this study, we investigated the impact of vitC pre-conditioning on the phenotype and functionality of CD19-targeting CAR-T cells (vitC-CAR19-Ts vs. CAR19-Ts) in co-culture with CD19+ Nalm-6 cells. We also generated CD19 knockout (CD19-) Nalm-6 cells using CRISPR/Cas9 as a negative control. Using multicolor flow cytometry, bulk-RNA transcriptomics, metabolic and multiplex assays, we analysed the activation and regulatory status as well as phenotypic characteristics and cytotoxic capacity of (vitC‑)CAR19-Ts during generation and upon target cell encounter.Results Strikingly, vitC-CAR19-Ts had a prominent effector memory phenotype and displayed increased long-term mitochondrial fitness when compared to CAR19-Ts. Moreover, vitC-CAR19-Ts showed significantly enhanced cytotoxicity towards CD19+ but not CD19- Nalm-6 cells. In line, their granulysin release was significantly increased, which was confirmed to originate from both CD4+ and CD8+ T cells by intracellular staining. RNA sequencing of (vitC-)CAR19-Ts after target cell encounter revealed upregulation of GNLY (granulysin) as well as genes involved in T-cell activation, metabolism and epigenetic reprograming. Furthermore, vitC-CAR19-Ts showed increased cytotoxicity upon repetitive target cell encounter when compared to CAR19-Ts.Conclusion In conclusion, we could show in this proof-of-principle study that vitC pre-conditioning leads to CAR19-T-cell products with significantly enhanced cytotoxicity and fitness, which is potentially epigenetically imprinted. Therefore, vitC pre-conditioning is a promising strategy to generate improved T-cell products with superior long-term cytotoxic and persistence capacity.Offenlegung Interessenkonflikt: The authors declare no conflict of interest.Invited talks abstract/summary

Evaluation of migratory and phagocytic properties of monocyte-derived dendritic cells from human umbilical cord blood

Background Umbilical cord blood (UCB) represents a unique source for stem cell transplantation while also harboring professional antigen-presenting cells such as monocytes. These may serve as a future source for monocyte-derived dendritic cells (MoDC) for clinical studies, yet the immunological properties of UCB-MoDC are still mostly unknown. This project aims to compare the functional properties of adult- and UCB-MoDC in terms of their migratory and phagocytic behavior.Methods Monocytes from adult healthy donors and UCB were magnetically enriched from PBMCs followed by their differentiation and maturation into MoDC. To assess endocytosis and pinocytosis, differentiated MoDC were exposed to FITC-Albumin and TITC-Dextran for 1 hour. Antigen uptake was evaluated by flow cytometry. Control groups were primed with respective inhibitors prior to antigen exposure. Migratory behavior was evaluated in the presence or absence of chemokines (MIP-3β, MIP-1α, SDF-1β). Maturated adult- and UCB-MoDC were added to 8µm transwell inserts, placed into well plates containing media with chemokines and incubated for 90 minutes at 37°C. Migrated MoDC were thereafter enumerated using a flow cytometer.Results UCB-MoDC presented macrophage mannose receptor (MMR)-mediated endocytosis of FITC-Dextran in comparison to controls, similar to adult MoDC in a concentration-dependent manner. UCB-MoDC sensitivity to MMR-blocking was similar to adult MoDC. Likewise, macropinocytosis-driven uptake of TITC-Albumin was unaltered in UCB-MoDC in comparison to their adult counterpart. Receptiveness to imipramine, an inhibitor of membrane ruffle formation, was similar between UCB- and adult MoDC. UCB-MoDC presented slightly elevated rates of MIP-3β- and SDF-1β-mediated chemotaxis. However, no significant differences could be detected in comparison to adult MoDC in terms of CCR7 (MIP-3β), CCR5 (MIP-1α) and CXCR4 (SDF-1β)-mediated transmigration.Conclusion UCB-derived MoDC present no significant differences compared to APB-derived MoDC in terms of antigen uptake and migratory behavior. These findings have important implications for the evaluation of UCB-MoDC suitability for future clinical applications.Offenlegung Interessenkonflikt: The authors declare no conflict of interest.

A protein corona around human platelet-derived EVs promotes regenerative functions

Background Human platelet lysate (hPL) is currently used as efficient substitute for fetal bovine serum for clinical cell manufacturing. Furthermore, we have previoulsy observed accelerated skin organoid formation and in vivo wound healing by hPL. As shown by others and us, EVs bear a biologically active protein corona, depending on the mode of preparation and the protein milieu.Methods In this study we asked whether hPL-derived EVs or platelet-derived soluble factors mediate these trophic effects of hPL. We separated EVs from soluble factors of hPL to understand the mode of action during skin organoid formation and immunomodulation as model systems for tissue regeneration. EVs were concentrated from hPL by tangential-flow filtration (TFF-EVs) and further purified by size-exclusion chromatography (TSEC-EVs) separating EVs from (lipo-) protein-rich soluble factors (TSEC-sol.F). Samples were characterized by tunable resistive pulse sensing, western blot, tandem mass-tag proteomics and super-resolution microscopy, and functionally tested during organoid formation and immunomodulation.Results We identified three major protein clusters by proteomic principle component analysis separating TSEC-EVs from hPL clustering with TFF-sol.F and TFF-EVs clustering with TSEC-sol.F. TFF-EVs induced significantly improved skin-organoid formation and inhibition of T-cell proliferation, compared to TSEC-EVs or to TSEC-sol.F. Reconstituting the corona on TSEC-EVs with TSEC-sol.F re-established functionality comparable to TFF-EVs. Zeta potential and super-resolution imaging confirmed corona formation.Conclusion TFF is a permissive technology enabling scalable enrichment and separation of functional corona-bearing EVs and soluble factors. Depletion of the TFF-EV corona by SEC or ultracentrifugation abrogated functionality indicating a novel mode of action. The corona could be artificially reconstituted in add-back of sol.F showing similar effects compared to TFF-EVs. This enables EV engineering with selected corona proteins for specific purposes and therapeutic applications.Offenlegung Interessenkonflikt: Kein Interessenskonflikt

Stem Cells

Overcoming immune escape in EBV-associated PTLD by understanding the two levels of immunosuppression: the tumor microenvironment and iatrogenic immunosuppression

Background Epstein-Barr virus (EBV) is one of the most common opportunistic pathogens causing morbidity in immunocompromised patients including the development of B-cell lymphomas (e.g. post-transplant lymphoproliferative disease, PTLD). Viral clearance requires the restoration or establishment of an EBV-specific T-cell response, which is impaired in PTLD patients due to the required immunosuppressive treatment (e.g. Tacrolimus, Tac) as well as the tumor microenvironment (TME).Methods Serving as in vitro EBV-associated PTLD model, EBV-transformed B-lymphoblastoid cell lines (B-LCLs) were established from &gt;15 healthy EBV-seropositive donors. At different time points of B-LCL generation, their immunomodulatory receptor expression and secretome were analyzed by flow cytometry and multiplex assays. B-LCL-conditioned medium (CM) was collected at selected time points and pooled from 8-12 donors to generate CMearly (B-LCL age 6-8 weeks), CMint (12-14 weeks) and CMlate (&gt;30 weeks) which were used to mimic the TME of PTLD in T-cell assays. EBV-specific memory T cells were expanded in presence of autologous B-LCLs and the effect of CM or Tac on their phenotype, activation level and functionality was analyzed by flow cytometry.Results Longitudinal analysis of immunomodulatory receptor expression by B-LCLs revealed significant upregulation of CD40, CD86 and PD-L1. Cytokine profile analysis revealed a remarkably high inter-individual variability. While the secretion of the pro-inflammatory IL-6 and IL-8 decreased significantly during B-LCL generation, IL-10 secretion was increased in a fraction of intermediate-aged B-LCLs. Due to its known immunosuppressive effect, two different CMint were generated based on IL-10 concentration (CMint-hi, CMint-lo). In all co-cultures of CD3+ T cells with irradiated autologous B-LCLs, central memory T cells were the main active subset. Activation and proliferation were markedly inhibited in presence of Tac as well as CMint and CMlate.Conclusion Creating an immunosuppressive TME is a key mechanism of tumor immune escape. Our data indicate longitudinal changes in TME composition during tumor development. Moreover, T-cell activation and proliferation, both basal attributes of T-cell functionality, were reduced in presence of CM as well as Tac. Our results provide insights into how not only Tac but also the TME affects T-cell responses, thus supporting the development of therapeutic strategies to overcome tumor-mediated immune escape.Offenlegung Interessenkonflikt: The authors declare no conflict of interest.

Discovery of the human cytomegalovirus-specific peptide repertoire naturally processed and presented by infected human antigen presenting cells

Background Infection with or reactivation of human cytomegalovirus (HCMV) remains a clinically problem in immunocompromised patients mainly caused by insufficient T-cell functionality. Knowledge of viral targets is critical to improve monitoring of high-risk patients and to optimize antiviral T-cell therapy. We aimed to identify naturally presented HLA-A*11:01-restricted HCMV-derived T-cell epitopes from HCMV-infected professional antigen presenting cells to expand the spectrum of immunogenic targets.Methods Monocyte-derived and genetically-engineered induced dendritic cells (iDCs) were generated to provide a stable platform for soluble (s)HLA-A*11:01 production. After infection of sHLA-A*11:01-secreting cells with wild type HCMV or a mutant lacking known immune evasion molecules (US2-6+11), sHLA-A*11:01-bound peptides were isolated by w6/32 immunoaffinity chromatography followed by mass spectrometric analysis. Identified peptides were screened for their viral protein origin and binding strength to HLA-A*11:01 using established databases. The highest scoring candidates were selected for in vitro evaluation of immunogenicity, cytotoxicity, clinical relevance and suitability for immune monitoring.Results More than 50 naturally presented candidate peptides were identified. The immunogenicity of the 25 highest scoring HLA-A*11:01-restricted peptides was evaluated, with functional active CMV-specific T cells detected against five candidates in healthy CMV+ donors by IFN-γ-EliSpot. Their complex stability was demonstrated by in vitro peptide binding assays. Highly proliferative and cytotoxic memory T cells were detected after stimulation with the UL36-derived A11SAL and UL122-derived A11SVS peptides in healthy CMV+ donors and HCMV-infected patients. Surprisingly, A11SAL-specific memory T cells exhibited functional properties at levels comparable to those T cells against the known immunodominant pp65-derived A02NLV peptide.Conclusion Eliciting strong antiviral T-cell responses in healthy CMV+ donors as well as HCMV-infected patients, the A11SAL peptide was identified as a new major target of the anti-HCMV immune response. Newly identified HCMV peptides expand the repertoire of immunodominant targets and will improve strategies for identification of high-risk patients, and enhancing therapeutic options using HCMV-specific T cells.Offenlegung Interessenkonflikt: The authors declare no conflict of interest.

Immunotherapy 1 - Unengineered Cells

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