Notch Signaling Facilitates In Vitro Generation of Cross-Presenting Classical Dendritic Cells

Kirkling, Margaret E.; Cytlak, Urszula; Lau, Colleen M.; Lewis, Kanako L.; Resteu, Anastasia; Khodadadi‐Jamayran, Alireza; Siebel, Christian W.; Salmon, Hélène; Mérad, Miriam; Tsirigos, Aristotelis; Collin, Matthew; Bigley, Venetia; Reizis, Boris

doi:10.1016/j.celrep.2018.05.068

Cited by 158 publications

(175 citation statements)

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“…This may also explain why it has been difficult to reveal consistent B and erythroid potential from intrathymic progenitors (Hao et al, 2008;Weerkamp et al, 2006) . From our scRNAseq, we could only find convincing evidence for the in vivo intrathymic development of DC-lineage cells, possibly from immigrating HPCs that expand in response to Notch signaling, consistent with recent studies (Kirkling et al, 2018) . Particularly pDC development was evident through an IRF8 hi expressing GMP precursor that most likely develops in the thymus given their virtual absence in the blood.…”

Section: Discussionsupporting

confidence: 90%

Single cell profiling of immature human postnatal thymocytes resolves the complexity of intra-thymic lineage differentiation and thymus seeding precursors

Lavaert

Vandamme

et al. 2020

Preprint

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During postnatal life, thymopoiesis depends on the continuous colonization of the thymus by bone marrow derived hematopoietic progenitors that migrate through the bloodstream. In human, the nature of these thymus immigrants has remained unclear. Here, we employ single-cell RNA sequencing on approximately 70.000 CD34 + thymocytes to unravel the heterogeneity of the human immature postnatal thymocytes. Integration of bone marrow and peripheral blood precursors datasets identifies several putative thymus seeding precursors that display heterogeneity for currently used surface markers as revealed by CITEseq. Besides T cell precursors, we discover branches of intrathymic developing dendritic cells with predominantly plasmacytoid DCs. Trough trajectory inference, we delineate the transcriptional dynamics underlying early human T-lineage development from which we predict transcription factor modules that drive stage-specific steps of human T cell development. Thus, our work resolves the heterogeneity of thymus seeding precursors in human and reveals the molecular mechanisms that drive their in vivo cell fate.

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Section: Discussionsupporting

confidence: 90%

Single cell profiling of immature human postnatal thymocytes resolves the complexity of intra-thymic lineage differentiation and thymus seeding precursors

Lavaert

Vandamme

et al. 2020

Preprint

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“…Several protocols have been proposed for the in vitro differentiation of human cDCs from CD34 + HSPCs 7, 38–40, 48, 49 . In vitro differentiated cDC1s have been shown to fully recapitulate both the phenotype and function of circulating bona fide blood cDC1s 8, 39, 48, 49 including high levels of IRF8 expression and Batf3-dependency in vitro 13 , as well as IRF8-dependancy both in vivo 70 and in vit ro 48 . Conversely, several aspects have limited an exhaustive validation of in vitro generated CD1c + cDC2-like cells such as the expression of CD14 7 , the transcriptional alignment with monocyte-derived DCs (moDCs) 39 or the lack of a high-dimensional phenotypic characterization 48, 49 .…”

Section: Discussionmentioning

confidence: 99%

“…This is in line with the crucial role of FLT3L, engaging the Flt3 receptor tyrosine kinase 41–43 in controlling DC homeostasis both in mice 32, 34, 44, 45 and humans 10, 29, 46, 47 . Moreover, the activation of Notch signaling pathway has been shown to further improve the in vitro differentiation of both human and mouse cDC1s 48, 49 . Despite the successes in modeling cDC1 differentiation in vitro , CD1c + cells found in culture of CD34 + HSPCs either align poorly with bona fide blood circulating cDC2s 39 or were not extensively characterized 48, 49 .…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, the activation of Notch signaling pathway has been shown to further improve the in vitro differentiation of both human and mouse cDC1s 48, 49 . Despite the successes in modeling cDC1 differentiation in vitro , CD1c + cells found in culture of CD34 + HSPCs either align poorly with bona fide blood circulating cDC2s 39 or were not extensively characterized 48, 49 .…”

Section: Introductionmentioning

confidence: 99%

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Engineered niches support the development of human dendritic cells in humanized mice

Anselmi

Vaivode

Dutertre

et al. 2019

Preprint

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AbstractClassical dendritic cells (cDCs) are rare sentinel cells specialized in the regulation of adaptive immunity. Modeling cDC development is both crucial to study cDCs and harness their potential in immunotherapy. Here we addressed whether cDCs could differentiate in response to trophic cues delivered by mesenchymal components of the hematopoietic niche where they physiologically develop and maintain. We found that expression of the membrane bound form of human FLT3L and SCF together with CXCL12 in a bone marrow mesenchymal stromal cell line is sufficient to induce the contact-dependent specification of both type 1 and type 2 cDCs from CD34+ hematopoietic stem and progenitor cells (HSPCs). Engraftment of these engineered mesenchymal stromal cells (eMSCs) together with CD34+ HSPCs creates an in vivo synthetic niche in the dermis of immunodeficient mice. Cell-to-cell contact between HSPCs and stromal cells within these organoids drive the local specification of cDCs and CD123+AXL+CD327+ pre/AS-DCs. cDCs generated in vivo display higher levels of resemblance with human blood cDCs unattained by in vitro generated subsets. Altogether, eMSCs provide a novel and unique platform recapitulating the full spectrum of cDC subsets enabling their functional characterization in vivo.

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“…We will need to further explore the potency of this response to that of common adjuvants and with a mix of target antigens to fully assess the utility of Sbi-III-IV as a universal vaccine adjuvant. For instance, comparison of the action of Sbi-III-IV to the Glaxo-Smith-Kline’s adjuvant systems, particularly AS01 [58], or to MF59 [59] may be of key interest and recent approaches may provide ideal pre-clinical model systems to facilitate this [60, 61] before progression to clinical studies. The work is ongoing but the data herein demonstrate the initial proof of concept.…”

Section: Discussionmentioning

confidence: 99%

Utilisation of staphylococcal immune evasion protein Sbi as a novel vaccine adjuvant

Yang

Back

Graewert³

et al. 2018

Preprint

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42Co--ligation of the B cell antigen receptor with complement receptor 2 on B--cells 43 via a C3d--opsonised antigen complex significantly lowers the threshold required 44 for B cell activation. Consequently, fusions of antigens with C3d polymers have 45 shown great potential in vaccine design. However, these linear arrays of C3d 46 multimers do not mimic the natural opsonisation of antigens with C3d. Here we 47 investigate the potential of using the unique complement activating 48 characteristics of Staphylococcal immune--evasion protein Sbi to develop a 49 pro--vaccine approach that spontaneously coats antigens with C3 degradation 50 products in a natural way. We show that Sbi rapidly triggers the alternative 51 complement pathway through recruitment of complement regulators, forming a 52 tripartite complex that acts as a competitive antagonist of factor H, resulting in 53 enhanced complement consumption. These functional results are corroborated 54 by the structure of this complement activating Sbi--III--IV:C3d:FHR--1 complex. 55Finally, we demonstrate that Sbi, fused with Mycobacterium tuberculosis antigen 56 Ag85b, causes efficient opsonisation with C3 fragments, thereby enhancing the 57 immune response significantly beyond that of Ag85b alone, providing proof of 58 concept for our pro--vaccine approach. 59 antigens by C3d at a molecular level and do not always enhance immune 82 responses [13]. After activation of C3, C3b attaches directly to the antigen 83 surface via the reactive thioester on the convex face of the protein's thioester 84 domain (TED). In the presence of complement regulators (factor I (FI) and its 85 co--factors, such as factor H (FH) and CR1) this is rapidly converted to iC3b and 86 then to C3d, exposing the concave CR2 binding site of the TED fragment away 87 from the antigen surface [14]. It is likely that multiple iC3b/C3d molecules attach 88 to complex antigens/pathogen surfaces during the initial activation phases of 89 complement, creating high--avidity binding site for complement fragment 90 receptors. 91In the last two decades, structural biology has helped to unveil many of the 92 molecular aspects that are crucial for the activation and regulation of the 93 complement system. Most notable are the crystal structures of the central 94 complement component activation states, native C3 [15], activated C3b and 95 inactive C3c [16]. The structure of C3b in complex with factors B and D [17] 96 subsequently revealed a detailed view of the alternative pathway C3 convertase 97 assembly and its activation, leading to the amplified cleavage of C3 molecules 98 that result in opsonisation and clearance of microbial pathogens and host debris. 99 The covalent attachment of C3b to surfaces does not discriminate between self 100 or non--self surfaces and requires tight regulation to protect host surfaces. 101 Structures of C3b in complex with FH domains 1--4 [18] and domains 19--20 [19, 102 20] provided insights into protection of host cells [21] and demonstrated how 103 5 factor H--related prote...

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Notch Signaling Facilitates In Vitro Generation of Cross-Presenting Classical Dendritic Cells

Cited by 158 publications

References 53 publications

Single cell profiling of immature human postnatal thymocytes resolves the complexity of intra-thymic lineage differentiation and thymus seeding precursors

Single cell profiling of immature human postnatal thymocytes resolves the complexity of intra-thymic lineage differentiation and thymus seeding precursors

Engineered niches support the development of human dendritic cells in humanized mice

Utilisation of staphylococcal immune evasion protein Sbi as a novel vaccine adjuvant

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