Innate lymphoid cells (ILCs), discovered about a decade ago, are a heterogeneous and rare lymphocyte population. Functionally analogous to T cells, ILCs lack antigen-specific receptors but share lineage-defining transcription factors (TFs), chemokine receptors, and cytokine profiles with T lymphocytes. While extensively studied using flow cytometry and single-cell RNA sequencing and mass cytometry, providing insights into their functional classification, activation states, and developmental trajectories, the precise localization and interactions of ILCs within their tissue microenvironments remain unclear. Helper ILCs (ILC1, ILC2, and ILC3), characterized by CD45+CD127+ expression and lack of other lineage markers (Lin−), are predominantly tissue-resident cells, particularly abundant in barrier sites. Their microanatomical localization is crucial for understanding their roles in tissue integrity, inflammation, and repair. Conventional immunofluorescence microscopy is limited in the number of markers it can simultaneously detect. Multiplexed histology techniques, such as the one employed in this study, overcome this limitation, enabling comprehensive *in situ* analysis of ILCs and their microenvironments. This study aims to map the phenotype and localization of ILCs using multi-epitope ligand cartography (MELC), a high-throughput multiplexed microscopy technique combined with a custom computational analysis pipeline to identify and characterize CD127+ ILCs *in situ*, analyze their microenvironments, and define microanatomical and molecular fingerprints characteristic of ILC localization and function in various chronically inflamed tissues. The analysis pipeline is adaptable for the *in situ* analysis of other immune cell types and tissue niches.

The study acknowledges previous work characterizing ILCs using flow cytometry and single-cell approaches such as RNA sequencing and mass cytometry. These techniques have contributed significantly to understanding ILC functional classification, activation states, and developmental trajectories. However, a gap in knowledge remains regarding the precise localization of ILCs within tissues and their interactions with the surrounding microenvironment. Previous studies have highlighted the importance of understanding these microenvironmental interactions for various immune cell subsets, as tissue-associated features define tissue and organ function and influence health or disease. The authors note the development of multiplexed histology techniques, such as imaging mass cytometry (IMC), CODEX, and tissue-based cyclic immunofluorescence (t-CyCIF), which have addressed the limitations of conventional immunofluorescence in analyzing multiple markers simultaneously. However, they state that these techniques haven't been extensively applied to the *in situ* characterization of ILCs and their microenvironments. This study aims to fill this gap in knowledge.

The researchers employed multi-epitope ligand cartography (MELC), an automated multiplexed microscopy technique, to analyze human tonsil and colon samples. The MELC technique involves sequential staining and imaging cycles, allowing for the detection of numerous markers within a single tissue section. A panel of antibodies targeting various leukocyte subsets, stromal markers, and transcription factors were used for staining. After image acquisition, a custom computational analysis pipeline was used for image processing and data analysis. This pipeline included steps for image registration, normalization, and cell segmentation using a random forest algorithm in Ilastik followed by object recognition in CellProfiler. The algorithm was trained on one tonsil dataset and applied to the other datasets without retraining. Single-cell features were extracted from the segmented images, allowing for the quantification of mean fluorescence intensities (MFIs) for each marker per cell. Cells were then classified into different immune cell types based on their marker expression profiles using a thresholding approach (for comparison with clustering method). ILCs were defined as Lin−CD45+CD127+ cells, while other immune populations (B cells, plasma cells, T helper cells, cytotoxic T cells, myeloid cells, and endothelial cells) were identified using similar criteria. Spatial analysis of ILC microenvironments was conducted by defining a 10 µm radius around each ILC as an "ILC niche." The composition of cell types and stromal components within these niches was quantified. In addition to the thresholding-based classification, a less biased approach involving t-distributed stochastic neighbor embedding (t-SNE) clustering analysis was performed on the high-dimensional single-cell data to identify ILCs and other cell populations. The spatial distribution of the clustered cells was compared to the distribution obtained through the fluorescence thresholding approach. For the validation of IRF4 expression on ILC3s, flow cytometry analysis was also performed on sorted tonsillar ILCs. Microarray analysis was used to validate IRF4 transcriptional levels in sorted ILC3s compared to hematopoietic progenitor cell populations.

The study successfully identified and quantified rare Lin−CD45+CD127+ ILCs *in situ* within human tonsil tissue using a combination of MELC and a custom-developed computational analysis pipeline. The analysis revealed a specific microenvironment for ILCs, enriched in plasma cells, vessels, and fibronectin fibers. Approximately 70% of ILCs were found within 10 µm of vessels and nearly 80% within the same distance from fibronectin fibers. Spatial analysis indicated that ILCs accumulate in distinct microanatomical areas, specifically the subepithelial connective tissue septum, where plasma cells also reside. This area is characterized by high vascularization and a specific stromal composition, suggesting a shared niche for both cell types. Clustering analysis of the multiplexed histology data confirmed the findings from the fluorescence thresholding, providing independent validation of the ILC identification and characterization. Furthermore, the analysis revealed that the transcription factor IRF4 is expressed in a subpopulation of tonsillar ILC3s, a finding validated at both the transcriptional and protein levels using microarray and flow cytometry, respectively. Interestingly, some ILC3s expressed the plasma cell marker CD138, although this was not consistently confirmed by flow cytometry analysis, possibly due to technical limitations. The study extended its findings to human colon samples from patients with ulcerative colitis, demonstrating conserved spatial localization patterns for ILCs across different tissues. In the colon, ILCs were found in close proximity to fibronectin and collagen fibers, often coating CD31+ endothelial cells, supporting the concept of conserved stromal landmarks for ILC localization.

The findings of this study address the limited understanding of the precise localization and microenvironmental interactions of human ILCs in tissues. The use of MELC and the advanced computational analysis pipeline allowed the researchers to overcome the limitations of conventional immunofluorescence, enabling comprehensive *in situ* analysis of rare immune populations. The identification of IRF4 as a marker for tonsillar ILC3s provides a valuable tool for future studies investigating ILC biology. The shared niche between ILCs and plasma cells in the subepithelial connective tissue septum suggests potential functional interactions between these cell types. The conserved spatial localization patterns observed in both tonsil and colon samples suggest that the ILC microenvironment plays a crucial role in their function and survival. This work establishes a platform for studying ILC biology in a spatially resolved manner, improving our understanding of ILCs' role in tissue homeostasis and inflammation. Future studies should explore the functional implications of the observed spatial localization and interactions between ILCs and their microenvironment. Further investigations are needed to clarify the precise role of IRF4 in ILC3 function and to fully understand the expression of CD138 on ILCs.

This study presents a novel approach for the high-throughput, in-depth characterization of ILCs and their microenvironments, identifying IRF4 as a marker for tonsillar ILC3s and highlighting conserved stromal landmarks for ILC localization across tissues. The findings suggest the existence of defined tissue patterns constituting ILC niches, paving the way for future investigations into ILC biology, tissue homeostasis and inflammation. Future research could focus on expanding this approach to other tissues and inflammatory conditions, investigating the functional implications of the identified interactions between ILCs and their microenvironment, and further exploring the role of IRF4 in ILC3 biology.

The study's analysis is primarily two-dimensional (2D), potentially overlooking aspects of the ILC microenvironment present in the natural three-dimensional (3D) tissue structure. While the researchers attempted to address potential signal cross-contamination from adjacent cells in the analysis, this remains a potential limitation, particularly regarding the CD138 expression on ILCs, which requires further validation. The relatively small sample size for certain analyses could also affect the generalizability of the findings. Finally, the study focused on specific markers; the absence of other markers in the panel doesn't rule out their involvement in ILC localization and function.