Guidelines for visualization and analysis of DC in tissues using multiparameter fluorescence microscopy imaging methods

Bayerl, Felix; Bejarano, David Alejandro; Bertacchi, Giulia; Doffin, Anne-Claire; Gobbini, Elisa; Hubert, Margaux; Li, Lijian; Meiser, Philippa; Pedde, Anna-Marie; Posch, Wilfried; Müller, Luise; Schlitzer, Andreas; Schmitz, Marc; Schraml, Barbara U.; Uderhardt, Stefan; Valladeau‐Guilemond, Jenny; Wilflingseder, Doris; Zaderer, Viktoria; Böttcher, Jan

doi:10.1002/eji.202249923

Cited by 16 publications

(7 citation statements)

References 86 publications

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“…2A) , using DAPI staining to perform cell annotation, BDCA2 (also named CLEC4C/CD303) for pDC detection, DC-LAMP (LAMP3) for mature DCs and CD1a for LCs. The cDC1 subset was detected using XCR1, the specificity of which was previously validated using coupled staining of CLEC9A mRNA by in situ hybridization and XCR1 by immunofluorescence (27). As cDC2 represent a very heterogenous population with many markers shared with macrophages, we couldn’t include this subset in the present analysis as we would need at least ten markers to identify them properly.…”

Section: Resultsmentioning

confidence: 99%

Spatial architecture of CD8⁺T cells and DC subsets is critical for the response to immune checkpoint inhibitors in melanoma

Gobbini,

Hubert,

Doffin

et al. 2024

Preprint

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BackgroundDendritic cells (DCs) are promising targets for cancer immunotherapies owing to their central role in the initiation and the control of immune responses. Their functions encompass a wide range of mechanisms mediated by different DC subsets. Several studies have identified human tumor- associated DC (TA-DC) populations through limited marker-based technologies, such as immunostaining or flow cytometry. However, tumor infiltration, spatial organization and specific functions in response to immunotherapy of each DC subset remain to be defined.MethodsHere, we implemented a multiplexed immunofluorescence analysis pipeline coupled with bio-informatic analyses to decipher the tumor DC landscape and its spatial organization within melanoma patients’ lesions, and its association with patients’ response to immune checkpoint inhibitors (ICI). For this aim, we analyze a cohort of 41 advanced melanoma patients treated with anti- PD1 alone or associated with anti-CTLA4. Distance and cell network analyses were performed to gain further insight into the spatial organization of tumor-associated DCs. A Digital Spatial Profiling analysis further characterized ecosystem of tumor-infiltrating DCs.ResultsPlasmacytoid DCs (pDCs) were the most abundant DC population, followed by conventional cDC1 and mature DCs, present in equal proportions. In contrast to CD8+T cell frequency, and despite varying densities, all DC subsets were associated with a favorable response to ICI. Distance and cell network analyses demonstrated that tumor-infiltrating DCs were largely organized in dense areas with high homotypic connections, except for cDC1 that exhibited a more scattered distribution. We identified four patterns of ecosystems with distinct preferential interactions between DC subsets. Significantly, the proximity and interactions between CD8+T cells and cDC1 were positively associated with patients’ response to ICI.ConclusionsOur study unravels the complex spatial organization of DC subsets and their interactions in melanoma patient lesions, shedding light on their pivotal role in shaping the response to ICI. Our discoveries regarding the spatial arrangement of cDC1, especially with CD8+ T cells, provide valuable clues for improving immunotherapeutic strategies in melanoma patients.What is already known on this topicDendritic cells (DCs) are promising targets for cancer immunotherapies owing to their central role in the initiation and the control of immune responses. Although conventional type 1 dendritic cells (cDC1) were proposed to contribute to immunotherapy response, their precise functions and interactions with other immune populations in human cancers are largely unknown.What this study addsThis study provides a precise characterization of the spatial distribution and organization of tumor- infiltrating DCs in a large cohort of advanced melanoma patients, and in correlation with response to immunotherapy. While DCs are organized in dense areas with high homotypic connections, cDC1 exhibit a more scattered distribution and form heterotypic aggregates with other DC subsets. More importantly, a close connection between cDC1 and CD8 T cell is uniquely correlated with the patients’ response to immunotherapy.How this study might affect research, practice or policyThis study improves our understanding of CD8-DC spatial organization within the tumor microenvironment and will have a broad spectrum of implications in the design of anti-tumor immune-activating compounds and the design of biomarkers of response to immunotherapy for melanoma patients.

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Section: Resultsmentioning

confidence: 99%

Spatial architecture of CD8⁺T cells and DC subsets is critical for the response to immune checkpoint inhibitors in melanoma

Gobbini,

Hubert,

Doffin

et al. 2024

Preprint

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“…To detect, identify, and quantify PDAC-infiltrating B cell subsets, formalin-fixed paraffin-embedded (FFPE) tissue sections were stained on a Ventana Discovery Ultra platform (Ventana Medical Systems, Basel, Switzerland) as described previously ( 39 ). The tyramide signal amplification-based Opal technology (Akoya Biosciences, Marlborough, MA, USA) enables simultaneous imaging of up to six markers in addition to DAPI.…”

Section: Methodsmentioning

confidence: 99%

Neoadjuvant chemotherapy is associated with suppression of the B cell-centered immune landscape in pancreatic ductal adenocarcinoma

Rupp,

Dietsche,

Kießler

et al. 2024

Front. Immunol.

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Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed at advanced stages and associated with early distant metastasis and poor survival. Besides clinical factors, the tumor microenvironment (TME) emerged as a crucial determinant of patient survival and therapy response in many tumors, including PDAC. Thus, the presence of tumor-infiltrating lymphocytes and the formation of tertiary lymphoid structures (TLS) is associated with longer survival in PDAC. Although neoadjuvant therapy (NeoTx) has improved the management of locally advanced tumors, detailed insight into its effect on various TME components is limited. While a remodeling towards a proinflammatory state was reported for PDAC-infiltrating T cells, the effect of NeoTx on B cell subsets, including plasma cells, and TLS formation is widely unclear. We thus investigated the frequency, composition, and spatial distribution of PDAC-infiltrating B cells in primary resected (PR) versus neoadjuvant-treated patients using a novel multiplex immunohistochemistry panel. The NeoTx group displayed significantly lower frequencies of pan B cells, GC B cells, plasmablasts, and plasma cells, accompanied by a reduced abundance of TLS. This finding was supported by bulk RNA-sequencing analysis of an independent fresh frozen tissue cohort, which revealed that major B cell pathways were downregulated in the NeoTx group. We further observed that plasma cells frequently formed aggregates that localized close to TLS and that TLS+ patients displayed significantly higher plasma cell frequencies compared to TLS- patients in the PR group. Additionally, high densities of CD20+ intratumoral B cells were significantly associated with longer overall survival in the PR group. While CD20+ B cells held no prognostic value for NeoTx patients, an increased frequency of proliferating CD20+Ki67+ B cells emerged as an independent prognostic factor for longer survival in the NeoTx group. These results indicate that NeoTx differentially affects PDAC-infiltrating immune cells and may have detrimental effects on the existing B cell landscape and the formation of TLS. Gaining further insight into the underlying molecular mechanisms is crucial to overcome the intrinsic immunotherapy resistance of PDAC and develop novel strategies to improve the long-term outcome of PDAC patients.

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“…For surface staining, cells were incubated with 50 µl purified anti-mouse CD16/32/FcBlock for 10 min at 4 °C, as described previously 45 . Additional antibodies were then added in FACS buffer to a final volume of 100 µl at 4 °C for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Interleukin-10 enhances recruitment of immune cells in the neonatal mouse model of obstructive nephropathy

Wyczanska,

Thalmeier,

Keller

et al. 2024

Sci Rep

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Urinary tract obstruction during renal development leads to inflammation, leukocyte infiltration, tubular cell death, and interstitial fibrosis. Interleukin-10 (IL-10) is an anti-inflammatory cytokine, produced mainly by monocytes/macrophages and regulatory T-cells. IL-10 inhibits innate and adaptive immune responses. IL-10 has a protective role in the adult model of obstructive uropathy. However, its role in neonatal obstructive uropathy is still unclear which led us to study the role of IL-10 in neonatal mice with unilateral ureteral obstruction (UUO). UUO serves as a model for congenital obstructive nephropathies, a leading cause of kidney failure in children. Newborn Il-10−/− and C57BL/6 wildtype-mice (WT) were subjected to complete UUO or sham-operation on the 2nd day of life. Neonatal kidneys were harvested at day 3, 7, and 14 of life and analyzed for different leukocyte subpopulations by FACS, for cytokines and chemokines by Luminex assay and ELISA, and for inflammation, programmed cell death, and fibrosis by immunohistochemistry and western blot. Compared to WT mice, Il-10−/− mice showed reduced infiltration of neutrophils, CD11bhi cells, conventional type 1 dendritic cells, and T-cells following UUO. Il-10−/− mice with UUO also showed a reduction in pro-inflammatory cytokine and chemokine release compared to WT with UUO, mainly of IP-10, IL-1α, MIP-2α and IL-17A. In addition, Il-10−/− mice showed less necroptosis after UUO while the rate of apoptosis was not different. Finally, α-SMA and collagen abundance as readout for fibrosis were similar in Il-10−/− and WT with UUO. Surprisingly and in contrast to adult Il-10−/− mice undergoing UUO, neonatal Il-10−/− mice with UUO showed a reduced inflammatory response compared to respective WT control mice with UUO. Notably, long term changes such as renal fibrosis were not different between neonatal Il-10−/− and neonatal WT mice with UUO suggesting that IL-10 signaling is different in neonates and adults with UUO.

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Guidelines for visualization and analysis of DC in tissues using multiparameter fluorescence microscopy imaging methods

Cited by 16 publications

References 86 publications

Spatial architecture of CD8⁺T cells and DC subsets is critical for the response to immune checkpoint inhibitors in melanoma

Spatial architecture of CD8⁺T cells and DC subsets is critical for the response to immune checkpoint inhibitors in melanoma

Neoadjuvant chemotherapy is associated with suppression of the B cell-centered immune landscape in pancreatic ductal adenocarcinoma

Interleukin-10 enhances recruitment of immune cells in the neonatal mouse model of obstructive nephropathy

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Guidelines for visualization and analysis of DC in tissues using multiparameter fluorescence microscopy imaging methods

Cited by 16 publications

References 86 publications

Spatial architecture of CD8+T cells and DC subsets is critical for the response to immune checkpoint inhibitors in melanoma

Spatial architecture of CD8+T cells and DC subsets is critical for the response to immune checkpoint inhibitors in melanoma

Neoadjuvant chemotherapy is associated with suppression of the B cell-centered immune landscape in pancreatic ductal adenocarcinoma

Interleukin-10 enhances recruitment of immune cells in the neonatal mouse model of obstructive nephropathy

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Spatial architecture of CD8⁺T cells and DC subsets is critical for the response to immune checkpoint inhibitors in melanoma

Spatial architecture of CD8⁺T cells and DC subsets is critical for the response to immune checkpoint inhibitors in melanoma