OMIP‐086: Full spectrum flow cytometry for high‐dimensional immunophenotyping of mouse innate lymphoid cells

Abstract This 25‐parameter, 22‐color full spectrum flow cytometry panel was designed and optimized for the comprehensive enumeration and functional characterization of innate lymphoid cell (ILC) subsets in mouse tissues. The panel presented here allows the discrimination of ILC progenitors (ILCP), ILC1, ILC2, NCR+ ILC3, NCR− ILC3, CCR6+ lymphoid tissue‐inducer (LTi)‐like ILC3 and mature natural killer (NK) cell populations. Further characterization of ILC and NK cell functional profiles in response to stimulation is provided by the inclusion of subset‐specific cytokine markers, and proliferation markers. Development and optimization of this panel was performed on freshly isolated cells from adult BALB/c lungs and small intestine lamina propria, and ex vivo stimulation with phorbol 12‐myrisate 13‐acetate, ionomycin, and pro‐ILC activating cytokines.


| BACKGROUND
ILCs are a unique subset of innate effector cells enriched at mucosal surfaces, with diverse roles in host defense, tissue remodeling and repair, inflammation, and metabolic homeostasis [1]. Despite lacking rearranged antigen receptors, ILCs display remarkable homology with conventional T helper (Th) type 1 (Th1), Th2, and Th17 cells in regards to phenotype and function, and thus are similarly classified into ILC1, ILC2, and ILC3 subsets [2]. Moreover, bona fide ILC subsets have now been expanded to include both cytotoxic NK and lymphoid tissueinducer (LTi) cells, which are phenotypically and functionally similar to ILC1 and ILC3 respectively, yet exhibit distinct developmental trajectories [3]. However, it is increasingly recognized that ILC subsets are Similarities to other OMIPS: There are no published OMIPs to date characterizing ILC subsets in multiple mouse tissues. not fixed, and that these cells can exhibit significant plasticity depending on the local inflammatory milieu [4][5][6][7][8]. As such, ILCs demonstrate intra-subset phenotypic heterogeneity depending on their specific microenvironment [9,10], and deep immunophenotyping therefore requires a comprehensive array of phenotypic and functional markers to accurately capture this biological variation.
Although crucial in multiple biological settings, ILCs constitute a relatively minor population within both mouse and human lymphoid and non-lymphoid tissues and blood, comprising 1%-5% of CD45 + leukocytes [5,[11][12][13][14][15]. Due to this inherent scarcity, acquiring as much information as possible on a single-cell level is of upmost importance for accurate ILC discrimination. High resolution ILC characterization is further convoluted in tissues such as the lung and small intestine, where the intrinsically autofluorescent nature of the samples results in a heightened background noise-to-signal ratio. With this in mind, cellular characterization via full spectrum flow cytometry provides a technological advancement given its high parameter capabilities [16] combined with the capacity to extract cellular autofluorescence profiles, thus improving discrimination of rare populations compared to conventional flow cytometry [17]. As such, the full spectrum panel whereby PLZF + ILCPs are restricted to the generation of ILC1, ILC2, and ILC3, but not LTi or NK cells [22,23]. In addition to PLZF, ILCPs display high-level expression of Il18r1, and thus a requirement for IL-18 signaling through IL-18Rα for proliferation and differentiation [19].
Importantly, both ILCPs and mature ILC subset are dependent on canonical IL-7 signaling for development and function, with expression of the receptor (IL-7Rα) and consumption of IL-7 dramatically greater in ILCs than their T cell counterparts [24]. Continual differenti-  Table 2 for all appropriate lineage markers).
ILC1s are characterized by their constitutive expression of T-bet (encoded by Tbx21), which is central for their production of interferon (IFN)-γ and ensuing response to intracellular pathogens following IL-12 and IL-18 stimulation [18,25]. Owing to their similarities with cytotoxic NK cells, ILC1s express NKp46, however tissue-specific distinctions can be made between these two subsets via the preferential expression of CD49a and/or TRAIL by ILC1, in conjunction with the absence of CD11b and CD49b [3,18,25].
Moreover, while lung ILCs lack CD4 expression [50], intestinal CCR6 + LTi-like ILC3s can be sub-classified as CD4 + and CD4 À [51]. As such, the inclusion of CD4 within the lineage cocktail may influence the characterization of ILC3 subsets on a tissue-specific basis. ILC cytokine production and proliferation within the lung ( Figure 1F) and siLP ( Figure 1G) was assessed in response to subset-specific activation CD11b within the lineage cocktail enables the enumeration of conventional mature CD11b + NKp46 + T-bet + NK cells ( Figure 1H) [52], with additional characterization of lung tissue-resident (trNK) and circulating (cNK) populations on the basis of CD49a and CD49b expression [53] ( Figure 1H). Markers including KLRG1 [54] and RORγt [55], and cytokines IFN-γ, IL-5, IL-13 [56] and IL-22 [57] are all reportedly expressed by NK cell subsets based on maturation, tissue localization and disease state, further highlighting the diversity of this OMIP.
In summary, we present here the first full spectrum flow cytometry panel ( Table 2)

CONFLICT OF INTEREST
The author declare that no conflicts of interest exist.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/cyto.a.24702.

ETHICS STATEMENT
All mouse experiments were performed in accordance with the rec-