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Philippon, C., Tao, S., Clement, D., Haroun-Izquierdo, A., Kichula, K. M., Netskar, H., . . . Malmberg, K. J. (2023). Allelic variation of KIR and HLA tunes the cytolytic payload and determines functional hierarchy of NK cell repertoires. Blood Advances, 7(16), 4492-4504
Open this publication in new window or tab >>Allelic variation of KIR and HLA tunes the cytolytic payload and determines functional hierarchy of NK cell repertoires
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2023 (English)In: Blood Advances, ISSN 2473-9529 , E-ISSN 2473-9537, Vol. 7, no 16, p. 4492-4504Article in journal (Refereed) Published
Abstract [en]

The functionality of natural killer (NK) cells is tuned during education and is associated with remodeling of the lysosomal compartment. We hypothesized that genetic variation in killer cell immunoglobulin-like receptor (KIR) and HLA, which is known to influence the functional strength of NK cells, fine-tunes the payload of effector molecules stored in secretory lysosomes. To address this possibility, we performed a high-resolution analysis of KIR and HLA class I genes in 365 blood donors and linked genotypes to granzyme B loading and functional phenotypes. We found that granzyme B levels varied across individuals but were stable over time in each individual and genetically determined by allelic variation in HLA class I genes. A broad mapping of surface receptors and lysosomal effector molecules revealed that DNAM-1 and granzyme B levels served as robust metric of the functional state in NK cells. Variation in granzyme B levels at rest was tightly linked to the lytic hit and downstream killing of major histocompatibility complex-deficient target cells. Together, these data provide insights into how variation in genetically hardwired receptor pairs tunes the releasable granzyme B pool in NK cells, resulting in predictable hierarchies in global NK cell function.

Place, publisher, year, edition, pages
American Society of Hematology, 2023
National Category
Cell Biology
Identifiers
urn:nbn:se:kth:diva-336311 (URN)10.1182/bloodadvances.2023009827 (DOI)001062192600001 ()37327114 (PubMedID)2-s2.0-85169323160 (Scopus ID)
Note

QC 20230912

Available from: 2023-09-12 Created: 2023-09-12 Last updated: 2023-09-22Bibliographically approved
Kienzle, T., Zambarda, C., Nasi, A., Önfelt, B. & Kadri, N. (2023). Characterization of interactions between monocytes and mesenchymal stromal cells. Cytotherapy, 25(6), S63-S63
Open this publication in new window or tab >>Characterization of interactions between monocytes and mesenchymal stromal cells
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2023 (English)In: Cytotherapy, ISSN 1465-3249, E-ISSN 1477-2566, Vol. 25, no 6, p. S63-S63Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Elsevier BV, 2023
Keywords
Desidual Basalis Mesenchymal Stromal cells, phagocytosis, monocytes
National Category
Cell Biology
Identifiers
urn:nbn:se:kth:diva-338229 (URN)001041036100128 ()
Note

QC 20231017

Available from: 2023-10-17 Created: 2023-10-17 Last updated: 2023-10-17Bibliographically approved
Carannante, V., Sandström, N., Olofsson, K., van Ooijen, H., Hell, B., Wiklund, M. & Önfelt, B. (2023). Generation of tumor spheroids in microwells to study NK cell cytotoxicity, infiltration and phenotype. In: Methods in Cell Biology: (pp. 195-208). Elsevier BV, 178
Open this publication in new window or tab >>Generation of tumor spheroids in microwells to study NK cell cytotoxicity, infiltration and phenotype
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2023 (English)In: Methods in Cell Biology, Elsevier BV , 2023, Vol. 178, p. 195-208Chapter in book (Other academic)
Abstract [en]

The development of new immunotherapeutic drugs and combinatorial strategies requires the implementation of novel methods to test their efficacy in vitro. Here, we present a series of miniaturized in vitro assays to assess immune cell cytotoxic activity, infiltration, and phenotype in renal carcinoma spheroids with the use of a recently developed multichambered microwell chip. We provide protocols for tumor spheroid formation, NK cell culture, fluorescence labelling and imaging of live or fixed cells directly in the chip together with data analysis.

Place, publisher, year, edition, pages
Elsevier BV, 2023
Keywords
3D killing assays, Infiltration, NK cells, Spheroids
National Category
Cell and Molecular Biology Cancer and Oncology
Identifiers
urn:nbn:se:kth:diva-338442 (URN)10.1016/bs.mcb.2023.01.001 (DOI)37516526 (PubMedID)2-s2.0-85148707250 (Scopus ID)
Note

Part of ISBN 9780443191633

QC 20231116

Available from: 2023-11-16 Created: 2023-11-16 Last updated: 2023-11-16Bibliographically approved
Carannante, V., Wiklund, M. & Önfelt, B. (2023). In vitro models to study natural killer cell dynamics in the tumor microenvironment. Frontiers in Immunology, 14, Article ID 1135148.
Open this publication in new window or tab >>In vitro models to study natural killer cell dynamics in the tumor microenvironment
2023 (English)In: Frontiers in Immunology, E-ISSN 1664-3224, Vol. 14, article id 1135148Article, review/survey (Refereed) Published
Abstract [en]

Immunotherapy is revolutionizing cancer therapy. The rapid development of new immunotherapeutic strategies to treat solid tumors is posing new challenges for preclinical research, demanding novel in vitro methods to test treatments. Such methods should meet specific requirements, such as enabling the evaluation of immune cell responses like cytotoxicity or cytokine release, and infiltration into the tumor microenvironment using cancer models representative of the original disease. They should allow high-throughput and high-content analysis, to evaluate the efficacy of treatments and understand immune-evasion processes to facilitate development of new therapeutic targets. Ideally, they should be suitable for personalized immunotherapy testing, providing information for patient stratification. Consequently, the application of in vitro 3-dimensional (3D) cell culture models, such as tumor spheroids and organoids, is rapidly expanding in the immunotherapeutic field, coupled with the development of novel imaging-based techniques and -omic analysis. In this paper, we review the recent advances in the development of in vitro 3D platforms applied to natural killer (NK) cell-based cancer immunotherapy studies, highlighting the benefits and limitations of the current methods, and discuss new concepts and future directions of the field.

Place, publisher, year, edition, pages
Frontiers Media SA, 2023
Keywords
NK cells, tumor microenvironment, tumor spheroids, tumor organoids, microscopy, flow cytometry, tissue sectioning, live cell imaging
National Category
Immunology in the medical area Cancer and Oncology
Identifiers
urn:nbn:se:kth:diva-333553 (URN)10.3389/fimmu.2023.1135148 (DOI)001027979300001 ()37457703 (PubMedID)2-s2.0-85164870765 (Scopus ID)
Note

QC 20230803

Available from: 2023-08-03 Created: 2023-08-03 Last updated: 2024-01-17Bibliographically approved
Sandoz, P., Kuhnigk, K., Szabo, E. K., Thunberg, S., Erikson, E., Sandström, N., . . . et al., . (2023). Modulation of lytic molecules restrain serial killing in γδ T lymphocytes. Nature Communications, 14(1), Article ID 6035.
Open this publication in new window or tab >>Modulation of lytic molecules restrain serial killing in γδ T lymphocytes
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2023 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 14, no 1, article id 6035Article in journal (Refereed) Published
Abstract [en]

γδ T cells play a pivotal role in protection against various types of infections and tumours, from early childhood on and throughout life. They consist of several subsets characterised by adaptive and innate-like functions, with Vγ9Vδ2 being the largest subset in human peripheral blood. Although these cells show signs of cytotoxicity, their modus operandi remains poorly understood. Here we explore, using live single-cell imaging, the cytotoxic functions of γδ T cells upon interactions with tumour target cells with high temporal and spatial resolution. While γδ T cell killing is dominated by degranulation, the availability of lytic molecules appears tightly regulated in time and space. In particular, the limited co-occurrence of granzyme B and perforin restrains serial killing of tumour cells by γδ T cells. Thus, our data provide new insights into the cytotoxic arsenal and functions of γδ T cells, which may guide the development of more efficient γδ T cell based adoptive immunotherapies.

Place, publisher, year, edition, pages
Springer Nature, 2023
National Category
Cell and Molecular Biology Cancer and Oncology
Identifiers
urn:nbn:se:kth:diva-338405 (URN)10.1038/s41467-023-41634-7 (DOI)001095471200022 ()37758698 (PubMedID)2-s2.0-85172238386 (Scopus ID)
Note

QC 20231123

Available from: 2023-10-23 Created: 2023-10-23 Last updated: 2023-12-04Bibliographically approved
Arruda, L. C. M., Stikvoort, A., Lambert, M., Jin, L., Sanchez-Rivera, L., Alves, R. M. P., . . . Uhlin, M. (2022). A novel CD34-specific T-cell engager efficiently depletes acute myeloid leukemia and leukemic stem cells in vitro and in vivo. Haematologica, 107(8), 1786-1795
Open this publication in new window or tab >>A novel CD34-specific T-cell engager efficiently depletes acute myeloid leukemia and leukemic stem cells in vitro and in vivo
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2022 (English)In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 107, no 8, p. 1786-1795Article in journal (Refereed) Published
Abstract [en]

Less than a third of patients with acute myeloid leukemia (AML) are cured by chemotherapy and/or hematopoietic stem cell transplantation, highlighting the need to develop more efficient drugs. The low efficacy of standard treatments is associated with inadequate depletion of CD34(+) blasts and leukemic stem cells, the latter a drug-resistant subpopulation of leukemia cells characterized by the CD34(+)CD38(-) phenotype. To target these drug-resistant primitive leukemic cells better, we have designed a CD34/CD3 bi-specific T-cell engager (BTE) and characterized its anti-leukemia potential in vitro, ex vivo and in vivo. Our results show that this CD34-specific BTE induces CD34-dependent T-cell activation and subsequent leukemia cell killing in a dose-dependent manner, further corroborated by enhanced T-cell-mediated killing at the singlecell level. Additionally, the BTE triggered efficient T-cell-mediated depletion of CD34(+) hematopoietic stem cells from peripheral blood stem cell grafts and CD34(+) blasts from AML patients. Using a humanized AML xenograft model, we confirmed that the CD34-specific BTE had in vivo efficacy by depleting CD34(+) blasts and leukemic stem cells without side effects. Taken together, these data demonstrate that the CD34-specific BTE has robust antitumor effects, supporting development of a novel treatment modality with the aim of improving outcomes of patients with AML and myelodysplastic syndromes.

Place, publisher, year, edition, pages
Ferrata Storti Foundation (Haematologica), 2022
National Category
Hematology Respiratory Medicine and Allergy Cancer and Oncology
Identifiers
urn:nbn:se:kth:diva-317010 (URN)10.3324/haematol.2021.279486 (DOI)000841120700009 ()35142149 (PubMedID)2-s2.0-85131531317 (Scopus ID)
Note

QC 20220906

Available from: 2022-09-06 Created: 2022-09-06 Last updated: 2022-09-06Bibliographically approved
Tay, A. H., Prieto-Diaz, R., Neo, S., Tong, L., Chen, X., Carannante, V., . . . Lundqvist, A. (2022). A(2B) adenosine receptor antagonists rescue lymphocyte activity in adenosine-producing patient-derived cancer models. Journal for ImmunoTherapy of Cancer, 10(5), e004592, Article ID e004592.
Open this publication in new window or tab >>A(2B) adenosine receptor antagonists rescue lymphocyte activity in adenosine-producing patient-derived cancer models
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2022 (English)In: Journal for ImmunoTherapy of Cancer, E-ISSN 2051-1426, Vol. 10, no 5, p. e004592-, article id e004592Article in journal (Refereed) Published
Abstract [en]

Background Adenosine is a metabolite that suppresses antitumor immune response of T and NK cells via extracellular binding to the two subtypes of adenosine-2 receptors, A(2)ARs. While blockade of the A(2A)ARs subtype effectively rescues lymphocyte activity, with four A(2A)AR antagonists currently in anticancer clinical trials, less is known for the therapeutic potential of the other A(2B)AR blockade within cancer immunotherapy. Recent studies suggest the formation of A(2A)AR/A(2B)AR dimers in tissues that coexpress the two receptor subtypes, where the A(2B)AR plays a dominant role, suggesting it as a promising target for cancer immunotherapy. Methods We report the synthesis and functional evaluation of five potent A(2B)AR antagonists and a dual A(2A)AR/A(2B)AR antagonist. The compounds were designed using previous pharmacological data assisted by modeling studies. Synthesis was developed using multicomponent approaches. Flow cytometry was used to evaluate the phenotype of T and NK cells on A(2B)AR antagonist treatment. Functional activity of T and NK cells was tested in patient-derived tumor spheroid models. Results We provide data for six novel small molecules: five A(2B)AR selective antagonists and a dual A(2A)AR/A(2B)AR antagonist. The growth of patient-derived breast cancer spheroids is prevented when treated with A(2B)AR antagonists. To elucidate if this depends on increased lymphocyte activity, immune cells proliferation, and cytokine production, lymphocyte infiltration was evaluated and compared with the potent A(2A)AR antagonist AZD-4635. We find that A(2B)AR antagonists rescue T and NK cell proliferation, IFN gamma and perforin production, and increase tumor infiltrating lymphocytes infiltration into tumor spheroids without altering the expression of adhesion molecules. Conclusions Our results demonstrate that A(2B)AR is a promising target in immunotherapy, identifying ISAM-R56A as the most potent candidate for A(2B)AR blockade. Inhibition of A(2B)AR signaling restores T cell function and proliferation. Furthermore, A(2B)AR and dual A(2A)AR/A(2B)AR antagonists showed similar or better results than A(2A)AR antagonist AZD-4635 reinforcing the idea of dominant role of the A(2B)AR in the regulation of the immune system.

Place, publisher, year, edition, pages
BMJ, 2022
Keywords
adenosine, immunotherapy, lymphocyte activation, lymphocytes, tumor-infiltrating
National Category
Cancer and Oncology Immunology in the medical area
Identifiers
urn:nbn:se:kth:diva-313312 (URN)10.1136/jitc-2022-004592 (DOI)000797594100003 ()35580926 (PubMedID)2-s2.0-85130152471 (Scopus ID)
Note

QC 20220602

Available from: 2022-06-02 Created: 2022-06-02 Last updated: 2023-06-08Bibliographically approved
Haroun-Izquierdo, A., Vincenti, M., Netskar, H., van Ooijen, H., Zhang, B., Bendzick, L., . . . Sohlberg, E. (2022). Adaptive single-KIR(+)NKG2C(+) NK cells expanded from select superdonors show potent missing-self reactivity and efficiently control HLA-mismatched acute myeloid leukemia. Journal for ImmunoTherapy of Cancer, 10(11), e005577, Article ID e005577.
Open this publication in new window or tab >>Adaptive single-KIR(+)NKG2C(+) NK cells expanded from select superdonors show potent missing-self reactivity and efficiently control HLA-mismatched acute myeloid leukemia
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2022 (English)In: Journal for ImmunoTherapy of Cancer, E-ISSN 2051-1426, Vol. 10, no 11, p. e005577-, article id e005577Article in journal (Refereed) Published
Abstract [en]

BackgroundNatural killer (NK) cells hold great promise as a source for allogeneic cell therapy against hematological malignancies, including acute myeloid leukemia (AML). Current treatments are hampered by variability in NK cell subset responses, a limitation which could be circumvented by specific expansion of highly potent single killer immunoglobulin-like receptor (KIR)(+)NKG2C(+) adaptive NK cells to maximize missing-self reactivity.MethodsWe developed a GMP-compliant protocol to expand adaptive NK cells from cryopreserved cells derived from select third-party superdonors, that is, donors harboring large adaptive NK cell subsets with desired KIR specificities at baseline. We studied the adaptive state of the cell product (ADAPT-NK) by flow cytometry and mass cytometry as well as cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq). We investigated the functional responses of ADAPT-NK cells against a wide range of tumor target cell lines and primary AML samples using flow cytometry and IncuCyte as well as in a mouse model of AML.ResultsADAPT-NK cells were >90% pure with a homogeneous expression of a single self-HLA specific KIR and expanded a median of 470-fold. The ADAPT-NK cells largely retained their adaptive transcriptional signature with activation of effector programs without signs of exhaustion. ADAPT-NK cells showed high degranulation capacity and efficient killing of HLA-C/KIR mismatched tumor cell lines as well as primary leukemic blasts from AML patients. Finally, the expanded adaptive NK cells had preserved robust antibody-dependent cellular cytotoxicity potential and combination of ADAPT-NK cells with an anti-CD16/IL-15/anti-CD33 tri-specific engager led to near-complete killing of resistant CD45(dim) blast subtypes.ConclusionsThese preclinical data demonstrate the feasibility of off-the-shelf therapy with a non-engineered, yet highly specific, NK cell population with full missing-self recognition capability.

Place, publisher, year, edition, pages
BMJ, 2022
Keywords
killer cells, natural, immunotherapy, adoptive, immunity, innate
National Category
Immunology in the medical area Cancer and Oncology
Identifiers
urn:nbn:se:kth:diva-322221 (URN)10.1136/jitc-2022-005577 (DOI)000883791000007 ()36319065 (PubMedID)2-s2.0-85141154858 (Scopus ID)
Note

QC 20221205

Available from: 2022-12-05 Created: 2022-12-05 Last updated: 2022-12-05Bibliographically approved
Hammer, Q., Perica, K., Mbofung, R. M., van Ooijen, H., Varady, E., Jelcic, M., . . . Malmberg, K.-J. (2022). Combined Genetic Ablation of CD54 and CD58 in CAR Engineered Cytotoxic Lymphocytes Effectively Averts Allogeneic Immune Cell Rejection. Paper presented at 64th Annual Meeting and Exposition of the American-Society-of-Hematology (ASH), DEC 10-13, 2022, New Orleans, LA. Blood, 140(Supplement 1), 1165-1166
Open this publication in new window or tab >>Combined Genetic Ablation of CD54 and CD58 in CAR Engineered Cytotoxic Lymphocytes Effectively Averts Allogeneic Immune Cell Rejection
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2022 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 140, no Supplement 1, p. 1165-1166Article in journal, Meeting abstract (Other academic) Published
Abstract [en]

Allogeneic cell therapies hold promise to be cost effective with scaled manufacturing for multi-dosing and on-demand off-the-shelf availability. A critical consideration for allogeneic cell products is their ability to persist, maintain function and avoid rejection by the patient's immune system. Genetic knockout (KO) of beta-2-microglobulin (B2M) leads to complete loss of cell-surface human leukocyte antigen (HLA) class I expression and efficiently abrogates CD8+ T-cell reactivity. However, loss of HLA class I triggers NK cell-mediated missing-self recognition and manipulation of B2M must therefore be combined with other immune-modulating strategies to limit recipient NK cell reactivity.We hypothesized that rejection by the patient's immune system can be diminished in primary CAR T cells, iPSC-derived T (iT) and NK (iNK) cells by reverse-engineering common tumor escape mechanisms. The adhesion molecules CD54 and CD58 are both present at the target cell side of the immune synapse, and loss of either of these molecules have previously been reported to elicit immune escape. Here, we show that the combined deletion of CD54 and CD58 in allogeneic immune effector cells makes them resistant to rejection by recipient immune cells through unidirectional reduced synapse formation (Figure 1A).HLA class I down-regulation by B2M silencing in primary T and NK cells triggered potent cytotoxicity by resting allogeneic NK cells. This response was mostly driven by educated NK cells expressing either NKG2A or killer cell immunoglobulin-like receptors (KIR) binding to HLA-E and HLA-C, respectively. However, over-expression of HLA-E or single HLA-C ligands in a K562 screening model only shut down the specific response of the NK cell subset carrying the cognate inhibitory receptor, resulting in only partial resistance to NK cells at the bulk level. Notably, the introduction of HLA-E was particularly detrimental in donors with expanded NKG2C+ NK cell subsets, due to its stimulatory effect through the activating NKG2C receptor. In contrast, combined deletion of CD54 and CD58 in target cells uniquely decreased the response of all tested NK cell subsets and showed universal reduction across NK cell populations from 18 healthy donors (Figure 1B). To delineate the mechanisms behind the increased resistance of target cells carrying these edits, we studied NK cell-target cell interactions at the single cell level by confocal microscopy in microchips. Allogeneic NK cells formed fewer conjugates and failed to form productive immune synapses with CD54-/-CD58-/- target cells, supporting the notion that they are more resistant to NK-cell mediated killing by unidirectional altered adhesion.We next introduced these edits in primary B2M-/- T cells engineered to express a second generation CAR19 from the TRAC locus. Corroborating the K562 screen, CD54-/-CD58-/-B2M-/- CAR-T cells had a selective survival advantage over B2M-/- CAR T cells and HLA-E-over-expressing B2M-/- CAR T cells in conventional mixed lymphocyte reaction (MLR) assays in vitro. Furthermore, we established an in vivo model to probe the effect of different genetic edits on the persistence of allogeneic cell therapy products. To this end, a mixed population of B2M-/- CAR T cells additionally bearing either CD54 and/or CD58 KO, HLA-E over-expression, or no further edits were infused into mice harboring allogeneic healthy donor PBMC. We found that CD54-/-CD58-/-B2M-/- CAR T cells had significantly better in vivo persistence compared to both B2M-/- CAR T cells and HLA-E+B2M-/- CAR T cells in the presence of PBMC from healthy donors (Figure 1B).Although multiplexed editing is feasible in primary CAR T cells, the iPSC platform has an unmatched capacity for homogenously introducing multiple immune-evasion strategies for off-the-shelf cell therapy. Similar to primary CAR T cells, multiplexed edited CD54-/-CD58-/-B2M-/-CIITA-/- iNK cells showed normal growth kinetics and were resistant to rejection by activated allogeneic NK cells in MLR assays.Together, these data demonstrate that reverse-engineering of common tumor escape mechanisms, which render target cells less susceptible to immune synapse formation, is an effective strategy to avert immune rejection of allogeneic CAR T and iPSC-derived CAR NK cells.

Place, publisher, year, edition, pages
American Society of Hematology, 2022
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:kth:diva-324747 (URN)10.1182/blood-2022-163086 (DOI)000893223201067 ()
Conference
64th Annual Meeting and Exposition of the American-Society-of-Hematology (ASH), DEC 10-13, 2022, New Orleans, LA
Note

QC 20230316

Available from: 2023-03-16 Created: 2023-03-16 Last updated: 2023-03-16Bibliographically approved
Hammer, Q., Perica, K., Mbofung, R. M., van Ooijen, H., Goodridge, J. P., Valamehr, B., . . . Malmberg, K. J. (2022). Genetic ablation of adhesion ligands effectively averts rejection of allogeneic immune cells. European Journal of Immunology, 52, 23-23
Open this publication in new window or tab >>Genetic ablation of adhesion ligands effectively averts rejection of allogeneic immune cells
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2022 (English)In: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 52, p. 23-23Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
WILEY, 2022
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:kth:diva-323909 (URN)000895436700020 ()
Note

QC 20230227

Available from: 2023-02-27 Created: 2023-02-27 Last updated: 2023-02-27Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0001-5178-7593

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