57. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

Prof. Dr. med.

Vorsitz

Prof. Dr.

&quot;New kid on the block&quot;: UL36-specific T cells contribute significantly to the HCMV-specific T-cell repertoire in healthy individuals

Immunotherapy and Gene Therapy

&quot;New kid on the block&quot;: UL36-spezifische T-Zellen tragen erheblich zum HCMV-spezifischen T-Zellrepertoire gesunder Menschen bei

Complications due to infection or reactivation with human cytomegalovirus (HCMV) remain a challenging problem in immunocompromised patients, mainly due to insufficient or absent T-cell immunity. Knowledge of immunodominant viral targets is crucial to classify T-cell responses to improve monitoring of high risk patients and optimize antiviral T-cell therapy. This study aims to evaluate whether the anti-apoptotic HCMV protein UL36 is suitable to expand the spectrum of immunogenic targets.Overlapping 15-mer peptides covering the whole UL36 protein were synthesized and assembled into one overlapping peptide pool (UL36pp) and 31 sub-pools containing 7─8 peptides (UL36sp1-31). UL36-specific T-cell responses were analyzed in comparison to the immunodominant HMCV peptide pools pp65pp and IE1pp in ≥40 healthy donors by IFN-γ-EliSpot assay. Peptides derived from UL36 sub-pools that elicited high T-cell responses were further evaluated for their binding strength to common HLA I and II alleles using established databases. Phenotypic and functional characteristics of UL36-specific T cells were assessed after antigen exposure by multicolor flow cytometry and multiplex assays to determine their immunogenicity and cytotoxic potential.Overall, high UL36-specific T-cell responses were detected in HCMV-seropositive donors. Surprisingly, more than 50% of all donors showed a stronger T-cell response to UL36pp (316.4 spw) than to pp65pp (244 spw). Compared to IE1pp (86.6 spw), the T-cell response was even stronger in 80% of donors. For strong responding UL36 sub-pools, we identified peptides with high binding scores covering a broad range of HLA alleles. In-depth profiling of phenotype and functionality of UL-36-specific memory T cells revealed an increased expression of activation markers (e.g. CD25, CD69) and a high proliferative and cytotoxic capacity after antigen contact. This was indicated by increased secretion of cytotoxic effector molecules (e.g. IFN-γ, granzyme B).Our results show that UL36 is a novel immunodominant target that exhibits an even stronger anti-HCMV T-cell immune response compared to the known responses against pp65 and IE1. Our data underline the impressive potential of UL36 in expanding the repertoire of immunogenic target antigens for improved monitoring of high-risk patients and optimization of adoptive antiviral T-cell therapy.The authors declare no conflict of interest.

Abstract lecture

Doxorubicin and anti-PD-L1 siRNA co-delivery with human heavy chain ferritin (HFn) nanoparticle for Acute myeloid leukemia (AML) treatment.

Doxorubicin und Anti-PD-L1 siRNA Abgabe mit HFn-Nanopartikeln zur Behandlung von akuter myeloischer Leukämie (AML)

Human heavy chain ferritin (HFn)-based drug delivery systems would significantly improve clinical efficacy and diminish potential side effects because of their unique structure, self-assembly properties, high biocompatibility, and CD71-mediated endocytosis. Anti-PD-L1 siRNA combination with Doxorubicin (DOX) can synergistically increase the release of tumor antigen and downregulate the level of PD-L1 on tumor cells, restoring the anti-tumor activity of T cells against Acute myeloid leukemia (AML). Therefore, this study aimed to produce the tumor-targeting DOX-siPD-L1/HFn nanocarrier for in vitro application in AML.The cDNA sequence of HFn was cloned into a pET-28a expression plasmid, and the constructed HFn/pET28a plasmid was subsequently transformed into host E. coli BL21 (Figure 1). After IPTG induction, HFn was purified with Ni-affinity chromatography and dialyzed against PBS. The purity and identity of recombinant HFn were analyzed using SDS-PAGE, western blot, and Dynamic light scattering. The final concentration was measured using the Bicinchoninic acid (BCA) assay. The encapsulation was performed using the standard pH system. The treatment effects of DOX-siPD-L1/HFn were carried out on HL-60 and K-562 cancer cell lines. The cytotoxicity of DOX-siPD-L1/HFn was measured using the MTT assay and Annexin V/PI staining, and flow cytometry and RT-PCR were used to determine the expression of PD-L1. The biocompatibility of DOX-siPD-L1/HFn has also been evaluated.Flow cytometry and RT-PCR analyses showed efficient siRNA-mediated PD-L1 silencing in AML cancer cell lines. The DOX-siPD-L1 co-delivery system showed higher cell proliferation arrest in K-562 cells compared to the liposome-encapsulated DOX, DOXIL. Besides, the apoptotic effects of DOX-siPD-L1/HFn on HL-60s were 3.42% and 2.77% higher than free and liposome-encapsulated DOX, respectively (Figure 2).Our study provided a promising strategy to combine DOX with PD-L1 silencing in AML. This efficient delivery system can also pave the way for the co-delivery of siRNAs and multiple chemotherapies to address the emerging needs of cancer combination therapy. The authors declare the lack of commercial or financial relationships that could be construed as a potential conflict of interest.

Compositional and functional analyses of ECP-elicited soluble factors

Zusammensetzungs- und Funktionsanalysen von ECP-ausgelösten löslichen Faktoren

Extracorporeal photopheresis (ECP) is a therapy for different T cell-mediated diseases that can exert both immune-stimulatory and immune-regulatory effects. However, its mechanism of action is poorly understood. Although previous studies have identified cellular mediators elicited by ECP, little is known about the implication of cell-free soluble components. Here, we studied the composition of ECP-elicited soluble factors and their functional effect on different immune cells in vitro.Human peripheral blood mononuclear cells (PBMCs) of healthy blood donors were treated with 8-methoxypsoralen (8-MOP) for 30 min and UVA in vitro. 12 different combinations of 8-MOP (0, 200, 300, or 400 ng/mL) and UVA (0, 2, or 3 J/cm2) were tested. Cell-free supernatants (ECP-SNs) were collected after 48h to measure cytokine and chemokine concentrations using a cytometric bead-based immunoassay. The impact of ECP-SNs on survival of immune cell subsets was tested by exposing PBMCs to ECP-SNs for 48h followed by Annexin V and Sytox staining by flow cytometry. The effect of ECP-SN on T cell proliferation and activation and was analyzed by stimulating magnetically enriched T cells with anti-CD3 in the presence or absence of ECP-SNs.ECP-SNs collected after 48h contained significantly higher levels of IL-6 and reduced levels of MCP-1 in an 8-MOP-dependent manner compared to untreated PBMCs. IL-1β and IFN-? were also increased and decreased respectively, though not significantly. IL-8, IL-10, IL-2, and TNF-α remained unchanged. Regarding the functional characterization of ECP-SNs, ECP-SNs did not affect survival of major immune cell subsets like B cells, T cells, monocytes, and NK cells upon 48h. Moreover, ECP-SNs did not alter T cell proliferation nor CD25, CD69, nor CD71 activation marker expression within 5 days.We showed that different ECP conditions can alter the composition of soluble factors that could be relevant to the mechanism of action of ECP. However, deeper compositional analyses such as metabolomics are needed. Moreover, this data suggests that soluble factors might not affect allogeneic T cell proliferation nor activation. Nevertheless, here we used ECP-SNs generated by unstimulated PBMCs that do not mimic inflammation present in patients. Further experiments testing ECP-SNs on additional immune cell subsets (e.g. monocytes and B cells) could unveil unknown mechanisms. This could help discover new biomarkers of potency of ECP and broaden its clinical application.There are no financial conflicts of interest to disclose

Prof. Dr. rer. nat.

Unraveling the Role of Galectins in Mesenchymal Stromal/Stem Cell-Mediated Immunomodulation through Extracellular Vesicles

Entschlüsselung der Rolle von Galektinen in der durch extrazelluläre Vesikel von mesenchymalen Stroma/Stammzellen vermittelte Immunmodulation

Mesenchymal stromal/stem cells (MSCs) hold immense therapeutic potentials due to their ability to modulate immune responses and promote regeneration, primarily through paracrine signaling, especially extracellular vesicles (EVs). The concrete mechanism of how MSC-EVs exert their therapeutic function remains spars.Galectins are crucial players in various physiological and pathological processes. By recognizing and bridging glycans on plasma membranes they have been shown to control various immune responses. Since selected galectins, especially galectins 1, 3, and 9, are expressed in MSCs and their function has been linked to EV biology, they might essentially contribute to the therapeutic function of MSC-EVs.In our effort in effectively translating MSC-EVs as a new proregenerative agent into the clinics, we have established clonally immortalized MSCs (ciMSCs). In addition to enable the scaled production of MSC-EV products for the clinical setting, these cells can be effectively genetically manipulated. Thus, they provide an ideal platform for dissecting the MSC-EVs" Mechanism of Action, which is crucial for developing surrogate methods providing insights into the therapeutic potency of the EV products. Here, we used the CRISPR/Cas-9 technology to delete the coding regions of the galectins 1, 3, and 9 in ciMSCs and explored the impacts of the deletion on biological properties of the cells and the immunomodulatory function of resulting EV products.In our ongoing experiments, deletion of galectin-1 did neither recognizably alter cell biological properties of the genetically engineered ciMSCs nor of their EV products. Currently, we explore impacts of the Galectin 3 and 9 knock outs. Moreover, the generation of various galectin knockout combinations will be explored in the future.The function of Galectin-1 is dispensable for the immunomodulatory properties of ciMSC-EV products that result in the reduction of the activated T cell pool within the mdMLR assay. Further experiments are required to finally determine the role of Galectins in the immunomodulatory potential of MSC-EVs and to understand whether and how they contribute to the mechanism of action of ciMSC-EVs.BG is a scientific advisory board member of Innovex Therapeutics SL, Mursla Ltd, ReNeuron Ltd. and PL BioScience. He is a founding director of Exosla Ltd. All other authors report no conflicts of interest.

The deletion of the immune checkpoint inhibitor receptor NKG2A with CRISPR/Cas9 improved the anti-leukemic efficacy of primary CD33-targeting CAR-NK cells

Der Knockout des Immun-Checkpoint-Inhibitor-Rezeptors NKG2A mit CRISPR/Cas9 verbessert die antileukämische Wirksamkeit von primären CD33-CAR NK Zellen

Chimeric antigen receptor (CAR) natural killer (NK) cells show safe and promissing clinical response and CAR33-NK cells demonstrated high antileukemic activity against acute myeloid leukemia (AML) in vivo (Albinger et al, BCJ, 2022). However, anti-tumor cytotoxicity is often impaired by tumor-mediated immune suppression (Bexte et al, OncoImmunology, 2022). Here, we report on a novel strategy to overcome CAR-NK cell inhibition by CRISPR/Cas9-based gene editing of the KLRC1-encoding gene encoding for the immune checkpoint NKG2A.Primary CD33-CAR NK cells were lentiviral transduction. KO of the KLRC1 locus was performed by CRISPR-Cas9 nucleofection. Genotyping of KLRC1-edited NK cells was performed using Inference of CRISPR Edits (ICE) analysis. To determine gene and surface marker expression we used single-cell level using CITE-seq. The in vivo-efficacy was evaluated in OCI-AML2 (GFP+, Luc+) xenografted NSG-SGM3 mouse models.The dual NK modification resulted in up to 60% CAR33-expressing cells, while KLRC1 gene disruption, quantified by insertion/deletion distribution profiles using Inference of CRISPR Edits (ICE) &gt;93%, resulted in 50% reduction of NKG2A cell surface expression. Single-cell multi-omics downstream analyses revealed a distinct gene regulation pattern in CAR33- and CAR33-KLRC1ko-NK cells with more mature and activated NK cells, which was preserved following exposure to AML cells. Importantly, CAR33-KLRC1ko-NK cells showed significantly higher elimination of AML cells compared to KLRC1ko-NK or CAR33-NK cells in vitro against AML-cell lines as well as against patient derived AML samples with high-risk subsets. In a NSG-SGM3 AML-xenograft mouse model, already a low dose injection of CAR33-KLRC1ko-NK cells led to a complete elimination of AML and leukemia-initiating cells in the bone marrow in vivo. The complete deletion of bone marrow AML cells was confirmed by bone marrow re-engraftment in vivo survival analysis.We conclude that dual modified NK cells have the potential to bypass immune suppression following contact with malignant cells not only in context of AML, but also in a broad range of other malignant diseases.None of the authors has COIs directly related to this manuscript. T.B, P.W. and E.U. have patent applications for gene-modified NK cell immune cell therapies for AML.

Abstract lecture (Abstract winner)

&shy;PSMA-specific natural killer cells for prostate cancer immunotherapy

Fließtext!Natural killer (NK) cells display built-in activity against tumor cells, which can be enhanced and rendered antigen-specific through expression of chimeric antigen receptors (CARs). Recently, CAR-expressing NK cells have been reported to confer promising safety and efficacy in patients with CD19-positive lymphoid neoplasms. However, CAR-NK cell therapy has shown no clear benefit thus far for treatment of solid tumors. Aim of this project is the generation and preclinical evaluation of CAR-NK cells for prostate cancer immunotherapy that recognize prostate-specific membrane antigen (PSMA). So far, we have developed robust and efficient protocols for the expansion and transduction of NK cells, as well as the functional evaluation of the resulting CAR-NK cells. Primary NK cells were derived from healthy donors and expanded using feeder cells. Transduction with a gamma-retroviral vector encoding a second-generation PSMA-specific CAR, IL-15 and the dLNGFR marker was embedded into the expansion process. Upon transduction, some 60% of NK cells expressed the transgenes over a period of several weeks, during which the CAR-NK cells maintained proliferative capacity. In order to assess anti-tumor activity in vitro, CAR-expressing NK cells were co-incubated with PSMA-positive or PSMA-negative tumor cells, and target cell killing was tracked over time using live cell imaging. Our results demonstrate that the CAR-NK cells readily targeted tumor cells in a CAR-dependent and PSMA-specific manner, which was further enhanced by IL-15. Based on these promising results, in vivo evaluation of the PSMA-specific CAR-NK cells in mouse tumor models is planned as the next step.xxxxxxxxxKeine

Immunotherapy

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