Were analyzed by using FlowJo computer software (Tree Star). Phenotypic analysis of human lymphocytes was performed using the following Abs reactive to human surface or intracellular antigens: eFluor780 Fixable Bone Morphogenetic Protein 1 Proteins Synonyms Viability Dye, APC-eFluor780 CD14 mAb (61D3), CD19 mAb (HIB19), CD3 mAb (SK7), CD123 mAb (6H6), eFluor660 anti-Eomes (WD1928), PE anti-T-bet (eBio4B10), or anti-GATA-3 (TWAJ), PE-Cy7 anti-Tbet (eBio4B10), APC anti-RORt (AFKJS-9) (eBioscience); APC – Vio770 CD141 mAb (AD54H12), anti-FcRI (CRA1), and CD11c mAb (MJ47G12), Fitc CD127 mAb (MB158C9), PEDazzle594 CD56 mAb (HCD56), PE-Vio770 NKp44 (2.29) (Miltenyi Biotec); BV605 CD117 mAb (104D2), PerCP-Cy 5.5 anti-CRTH2 (BM16), Pacific Blue CD94 mAb (XA185) (conjugated in house). four.5 Information analysis–How can we determine these IL-2R gamma/Common gamma-Chain Proteins medchemexpress various NK cell and ILC subsets in different tissues from distinct species The analysis of NK cells is described in the NK chapter by Moretta et al., where readers can uncover much more particulars (See also Chapter VI Section five Organic Killer cells). ILCs are present in diverse organs as tissue-resident cells but are also detected within the circulation [1346, 1352]. In mouse modest intestinal (SI) lamina propria (LP), all ILCs, namely NK cells, ILC1, ILC2, and ILC3 may very well be described [1345, 1353]. In Fig. 156 a gating method for murine ILCs derived from SI LP is shown; on the other hand, it needs to be stressed that ILC populations are usually not equally distributed in all organs and display some tissue-specific phenotypic differences. Combination of intranuclear staining of master transcription elements, namely T-bet (expressed on ILC1, NK cells and a subset of murineEur J Immunol. Author manuscript; obtainable in PMC 2020 July 10.Cossarizza et al.PageILC3), Eomes (NK cells), RORt (ILC3), and GATA3 (ILC2) collectively with NKp46 and CD127 (IL-7R) (Figure 156) or CD90 (not shown) enables identification of ILC subsets in all organs analyzed. Among SI LP CD45+ Lin- cells, NKp46 (or NK1.1) is often expressed not simply on NK cells but in addition on ILC1 in addition to a subset of ILC3. Therefore, staining of transcription elements is beneficial to dissect their identity (see also Chapter V Section 13 Transcription components). It has been proposed that SI LP NK cells is usually defined as NKp46+ RORt- T-bet+ Eomes+ cells, although ILC1 are NKp46+RORt- T-bet+ Eomes- cells [1345] (Figure 156). However, a population of cytotoxic NKp46+ RORt- T-bet+ Eomes+ intraepithelial ILC1 has been also described [1354]. In addition, the evaluation of NK/ILC1 in distinct mouse compartments revealed a higher degree of phenotypic and functional complexity [1346, 1355], suggesting that distinction involving NK and ILC1 cells might be extra difficult. ILC2 and ILC3 are enriched among SI LP CD45+ Lin- CD127+ lymphocytes and can be identified right after intranuclear staining of GATA3 and RORt as GATA3hi RORt- ILC2 and of GATA3lo RORt+ ILC3 (Figure 156) [1353, 1356]. Surface markers for example ST2 (IL-33R), CD25, ICOS, or KLRG1 have also been generally used to identify ILC2 [1353, 1357, 1358]. As previously described, expression of these markers slightly varies in different compartments. SI LP RORt+ ILC3 could be dissected into three main subsets based on NKp46 and CD4 expression (Figure 156), namely CD4+ ILC3, which functionally and phenotypically resemble fetal LTi and preferentially make IL-17 and IL-22; NKp46+ ILC3, which expand postnatally, co-express RORt and T-bet and create IL-22 and IFN-; and CD4- NKp46- ILC3, which basically represent a heterogeneous population of CCR6+ cells.
