The skin is our largest organ and serves as the primary barrier against infection and injury. Studies leveraging classical tools like histology and immunofluorescence have previously highlighted the spatial compartmentalization of immune surveillance in the skin across the epidermis, dermis, and subcutis. The epidermis is a region rich in Langerhans cells that migrate upon antigen uptake interspersed between keratinocytes, while the dermis contains dendritic cells, macrophages, mast cells, T cells, and innate lymphoid cells (ILCs) [1]. The subcutis also contains diverse immune cells and adipose cells with intrinsic immune function [1]. Recently, novel single-cell and ST tools have uncovered the cellular heterogeneity and complexity of the skin and have helped define multiple scales of heterogeneity in cutaneous immune organization.

In the immune landscape of the skin, distinct scales of heterogeneity are apparent, enabling compartmentalized immune functions. On the scale of individual immune cells, heterogeneity exists in the form of diverse cells and subpopulations, including canonical cells of innate and adaptive immunity as well as non-immune cells with increasing evidence of immune function such as vascular endothelial cells. On a larger scale, these cells organize into immunological niches that exhibit microanatomical heterogeneity, such as skin appendages like hair follicles and perivascular sites, which each exhibit unique immune cell organization. On the largest scale, macroanatomical heterogeneity encompasses variations in cell types and their spatial organization across body sites, which may be associated with distinct anatomic site predilections of skin disease [2]. scRNA-seq and ST are beginning to define the differences in skin composition across body sites, with paradigm-shifting findings for how we define cutaneous landscapes in different body sites.

Single-cell and spatially resolved technologies build upon traditional bulk transcriptomic profiling by allowing for the identification of specific cellular populations and their contributions to the immune landscape of the skin.

scRNA-seq has become a mainstay of biomedical research, advancing upon bulk RNA sequencing by allowing researchers to not only measure gene expression within tissues, but also allowing for the deconvolution of cell types. While bulk-RNA sequencing provides an average of gene expression of the whole tissue, scRNA-seq provides a more granular view, allowing for the measurement of genes in individual cells.

While scRNA-seq is well-established, more recently developed ST methods have enabled researchers to pinpoint the spatial localization of relevant cellular populations or gene expression programs in the skin [3]. These technologies fall into two major categories: 1) capture spot-based methods leveraging next-generation sequencing and 2) imaging-based transcript capturing techniques. First, spot-based technologies are those that allow for rapid profiling of tissues and capture the whole transcriptome using next-generation sequencing. Spot-based ST methods such as 10X Visium [4], slide-seq [5], and Stereo-seq [6] allow for the profiling of thousands of genes at once in 10–55 µm wide “spots” arranged in a grid fashion across the tissue. At the largest 55 µm spot size, 10X Visium is the only assay not reaching single-cell resolution. The newer 10X Visium HD platform reduced spot size to 2 µm in diameter, therefore enabling subcellular resolution analyses. Second, imaging-based methods include MERFISH [7], seqFISH [8], 10X Xenium [9], [10], and CosMx [11] which build on small molecule fluorescence in situ hybridization (smFISH) to profile a targeted panel of genes at single-cell or sub-cellular resolution [12]. In a trade-off for higher sensitivity transcript detection, these technologies use predefined gene target panels that do not cover the full transcriptome, although gene panels have rapidly increased in size (now up to ∼6000 genes for some assays). ST has undergone waves of technological innovation at a rapid pace that increasingly address limitations of each method, but each technology has distinct strengths and limitations in their current forms (Table 1). Optimized tissue processing protocols and improvements to downstream bioinformatics pipelines will likely facilitate even more detailed spatial analyses of cellular mediators of disease in the coming years (Fig. 1).

Single-cell and ST methods are being increasingly applied to experimental animal models to disentangle the molecular mechanisms of immune, epithelial, and stromal interactions within the skin. These efforts, combined with studies profiling the skin tissues of patients with diverse diseases ranging from inflammatory skin diseases to skin cancer, have provided clinically relevant insights into disease mechanisms that may inform patient risk stratification, predict treatment response, and identify novel therapeutic targets. The importance of the skin microbiome in additionally shaping cell-cell interactions in health and disease is well-established and beyond our current focus, and we refer readers to recent reviews on this topic [13], [14]. Aging, likewise, is an additional dimension for a comprehensive understanding of the cutaneous immune system, which has been covered in recent reviews [15], [16]. Here, we discuss recent efforts that leverage the latest technologies to characterize the immune landscape of the skin in homeostasis and disease at single-cell and spatial resolution in human samples and murine models.