Alsya J. Affandi

Cancer vaccines' efficacy to stimulate T cell responses in patients is currently sub-optimal, and this can be potentially overcome by the delivery of antigens directly to antigen-presenting cells (APCs). To achieve this, single domain antibodies (nanobodies, Nbs) specific for receptors on APCs can be conjugated to liposomes to deliver cancer antigens. Here, we generated three CD169-specific Nb liposomes containing 3 different Nb clones (1B5, 1C1, and 2C2) and one CLEC9A-specific Nb liposome. Blocking experiments with CD169-specific antibodies indicated distinct binding epitopes on CD169 for 2C2 compared with 1B5 and 1C1. Both 2C2 and 1C1 Nb liposomes showed stronger binding and uptake by CD169-overexpressing cell lines and monocyte derived dendritic cells compared to 1B5 liposomes. Analysis of primary blood-derived APCs demonstrated strong uptake of 2C2 and 1C1 Nb liposomes by Axl+ Siglec-6+ DCs, classical monocytes, and DC3s, whereas CLEC9A-specific Nb liposomes were taken up by CLEC9A-expressing XCR1+ DC1s. In contrast, when gp100-specific T cell activation was evaluated using gp100-containing CD169-specific Nb liposomes and moDCs, only 1B5 Nb liposomes led to an enhanced T cell response. Using enriched blood DCs, we also observed an increased T cell activation by gp100-containing CLEC9A-specific Nb liposomes. To conclude, CD169- and CLEC9A-specific Nb liposomes are specifically taken up by primary CD169- or CLEC9A-expressing human APCs, and, depending on the Nb, can lead to a higher T cell activation. These results highlight the potential of Nb liposomes to improve vaccination strategies for immunotherapy.

Dendritic cells (DCs) orchestrate immune responses. Their development is controlled by transcription factors, but epigenetic mechanisms remain poorly understood. DOT1L emerges as a key epigenetic regulator in immune cells. Mapping DOT1L-mediated histone H3K79 methylation in canonical DC subsets revealed that DOT1L modified common and DC subset–specific genes. Deletion of Dot1l in vivo or in vitro decreased myeloid progenitors and increased cDC2s, whereas cDC1s remained unchanged. In addition, in vitro deletion of Dot1l led to loss of plasmacytoid DCs (pDCs) and of IFNα production upon stimulation. Upon in vivo deletion, a decrease in pDCs was only observed after subsequent in vitro expansion. This difference was likely related to insufficient replication-mediated loss of H3K79 methylation in vivo within the time frame studied. Transcriptomes of Dot1l -KO DC subsets exhibited enrichment of antigen presentation pathways, and MHC class II surface levels were up-regulated in pDCs. Mechanistically, inhibition of DOT1L linked the observed effects to its methyltransferase activity. Together, our data indicate that in DCs DOT1L differentially affects the development of canonical subsets and suppresses antigen presentation pathways.

Figure: Background: Siglec-1 (Sialoadhesin/CD169) is expressed on myeloid cells and plays a key role in host defences by capturing incoming sialylated-pathogens such as Campylobacter jejuni . However, binding to Siglec-1 has also been exploited by pathogens such as SARS-CoV-2 for further dissemination. Results: Here we identified high-affinity VHHs also known as single-domain antibodies or Nanobodies that bind to Siglec-1 and allo- or orthosterically modulate ligand binding. VHH 2C2 was shown to bind directly to the ligand binding site of Siglec-1 and blocked binding of ganglioside liposomes and Campylobacter jejuni to monocyte-derived dendritic cells (moDCs) and ex vivo Siglec-1 ⁺ DCs. VHH 2C2 also blocked SARS-CoV-2 binding of moDCs. In contrast, the VHHs 1B5 and 1C1 interacted with Siglec-1 outside the ligand binding site and acted as positive allosteric modulators of Siglec-1 ligand interactions, as was illustrated by increased ganglioside liposome and Campylobacter jejuni binding by moDCs. Our data suggests that mechanistically, the VHH 1B5 and 1C1 interfere with the cis -binding sialic acids present on the Siglec-1-expressing cell and thereby enhance trans -interactions with ligands. Conclusion: In conclusion, we have isolated VHH that enhance or block Siglec-1 ligand binding to a variety of sialylated-pathogens enabling further interrogation of Siglec-1 function. Moreover, unlike conventional blocking antibodies targeting specific pathogens, Siglec-1 binding VHH could potentially serve as broad-spectrum pathogen blocking agents.

Dendritic cells (DCs) are important orchestrators of immune responses. Their development in the bone marrow is controlled by transcription factors, but epigenetic mechanisms remain poorly understood. DOT1L is emerging as a key epigenetic regulator in immune cells. By mapping DOT1L-mediated histone H3K79 methylation in canonical DC subsets, we observed that DOT1L modified common as well as DC subset-specific genes. In vitro- or in vivo- induced deletion of Dot1l followed by in vitro cell culture resulted in a decrease in myeloid progenitors and plasmacytoid DCs (pDCs) and an increase in cDC2s, while cDC1s remained unchanged. In vitro generated Dot1l- KO DCs were unable to produce IFNα upon stimulation. Moreover, transcriptomes of Dot1l -KO DC subsets exhibited enrichment of antigen presentation pathways and MHC class II surface levels were upregulated in pDCs. Mechanistically, inhibition of DOT1L linked the observed effects to its methyltransferase activity. Together, our data indicate that in DCs DOT1L differentially affects the development of canonical subsets and suppresses antigen presentation pathways.

Dendritic cells (DCs) are critical sentinels of the immune system, serving as indispensable bridges between innate and adaptive immune responses. DCs are a heterogeneous population, with subsets playing specialized roles in immune defense, tolerance, or disease development. Among these, the recently redefined DC3 subset has gained attention for its unique features and potential roles in health and disease. This review focuses on the phenotypic, functional, and developmental diversity of DC subsets—primarily DC3s—and their contributions to cancer. The tumor microenvironment (TME) in solid tumors is characterized by varying degrees of immune cell infiltration, including DCs. Within the TME, DCs play diverse roles, either promoting anti-tumor responses or facilitating immune evasion. Key subsets include conventional type 1 and type 2 DCs (cDC1s and cDC2s), as well as plasmacytoid DCs (pDCs). DC3s share certain features with cDC2s and monocytes but are distinct in their phenotype, function, and ontogeny. Functionally, DC3s can prime and activate T cells, skewing CD4+ T cells towards Th17 and stimulating CD8+ T cells with a tissue-resident memory phenotype. In cancer, their presence correlates with diverse outcomes depending on the TME: DC3 presence is linked to increased survival in patients with pancreatic ductal adenocarcinoma and oropharyngeal cancer while in non-small-cell lung cancer and melanoma it is associated with immunosuppression. The emerging understanding of DC3s highlights the complexity of DC biology and its relevance to diseases. The dynamic immunomodulatory functions of DC3s open new avenues for developing targeted therapies against cancer and immune-mediated disorders.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis with a 5‐year survival of less than 10%. More knowledge of the immune response developed in patients with PDAC is pivotal to develop better combination immune therapies to improve clinical outcome. In this study, we used mass cytometry time‐of‐flight to undertake an in‐depth characterization of PBMCs from patients with PDAC and examine the differences with healthy controls and patients with benign diseases of the biliary system or pancreas. Peripheral blood mononuclear cells from patients with PDAC or benign disease are characterized by the increase of pro‐inflammatory cells, as CD86⁺ classical monocytes and memory T cells expressing CCR6⁺ and CXCR3⁺, associated with T helper 1 (Th1) and Th17 immune responses, respectively. However, PBMCs from patients with PDAC present also an increase of CD39⁺ regulatory T cells and CCR4⁺CCR6⁻CXCR3⁻ memory T cells, suggesting Th2 and regulatory responses. Concluding, our results show PDAC develops a multifaceted immunity, where a proinflammatory component is accompanied by regulatory responses, which could inhibit potential antitumor mechanisms.

Background: Human lymph node (HuLN) models have emerged with invaluable potential for immunological research and therapeutic application given their fundamental role in human health and disease. While fibroblastic reticular cells (FRCs) are instrumental to HuLN functioning, their inclusion and recognition of importance for organotypic in vitro lymphoid models remain limited. Methods: Here, we established an in vitro three-dimensional (3D) model in a collagen-fibrin hydrogel with primary FRCs and a dendritic cell (DC) cell line (MUTZ-3 DC). To study and characterise the cellular interactions seen in this 3D FRC-DC organotypic model compared to the native HuLN; flow cytometry, immunohistochemistry, immunofluorescence and cytokine/chemokine analysis were performed. Results: FRCs were pivotal for survival, proliferation and localisation of MUTZ-3 DCs. Additionally, we found that CD1a expression was absent on MUTZ-3 DCs that developed in the presence of FRCs during cytokine-induced MUTZ-3 DC differentiation, which was also seen with primary monocyte-derived DCs (moDCs). This phenotype resembled HuLN-resident DCs, which we detected in primary HuLNs, and these CD1a⁻ MUTZ-3 DCs induced T cell proliferation within a mixed leukocyte reaction (MLR), indicating a functional DC status. FRCs expressed podoplanin (PDPN), CD90 (Thy-1), CD146 (MCAM) and Gremlin-1, thereby resembling the DC supporting stromal cell subset identified in HuLNs. Conclusion: This 3D FRC-DC organotypic model highlights the influence and importance of FRCs for DC functioning in a more realistic HuLN microenvironment. As such, this work provides a starting point for the development of an in vitro HuLN.

Editorial: Novel Platform for Antigen Delivery to Dendritic Cells for Immunotherapy