In addition, ILC2 levels in the lung lymph nodes and spleen were found to be increased on day 7 (Supplemental Figures E4 CCD and E5 ACB). did not Phen-DC3 affect lung ILC2 levels. Conclusion ILC2s increase in number in the mouse lung not only through local proliferation, Phen-DC3 but also through trafficking from the circulation into the lung using 2 rather than 1 or 4 integrins. contamination19, suggesting that a trafficking defect may be involved, but whether ILC2-expressed adhesion receptors may account for this reduction in ILC2 levels in the lung was not examined. We hypothesized that bone marrow derived ILC2s migrating through the blood express adhesion molecules that strongly bind to counter-receptors expressed by lung endothelium during allergic inflammation. This firm adhesion of ILC2s to pulmonary endothelium then allows for their subsequent migration into the tissues through chemotactic mediators, such as prostaglandin D2. The most common method by which leukocytes (other than ILC2s) are known to migrate from the blood into tissues and specifically the airways is usually mediated by leukocyte-expressed selectins and integrins.20 Leukocyte-expressed STATI2 selectins, such as L-selectin (CD62L), mediate the first step of leukocyte tethering to endothelium induced lung inflammation. METHODS Human ILC2 Human ILC2s were identified by flow cytometry as CD45+Lin?CRTH2+ lymphoid cells8, 27 in single cell suspensions of either post mortem human lungs and lymph nodes, or human peripheral blood, obtained as described in the online supplement. Mouse ILC2: Airway administration of and blocking of integrins Mice were challenged intranasally with three times (days 0, 3, and 6) and were sacrificed one day after the final challenge (day 7) as previously described10 and detailed in the online supplement. In experiments involving blocking of integrins, mice were administered intraperitoneally an integrin specific blocking or control antibody (1, 2, 4, or L antibody) (see Supplement Table EII) prior to each challenge (days 0, 3, and 6) as detailed in the online supplement. ILC2 proliferation, apoptosis, and Th2 cytokines ILC2 proliferation (Ki-67 staining), apoptosis (annexin V staining) and Th2 cytokines (IL-5, IL-13 staining) were quantitated by flow cytometry as described in the online supplement. Statistical analysis Statistical analysis was performed using Prism software (Graphpad, La Jolla, CA) as described in the online supplement. 0.05 was considered statistically significant. RESULTS Human ILC2s express 1 and 2 integrins To determine whether human ILC2s express adhesion molecules commonly associated with migration of Phen-DC3 other leukocytes, human ILC2s derived from peripheral blood, lung tissue, and intrathoracic lymph nodes were analyzed for expression of adhesion molecules by flow cytometry. Human ILC2s were gated as CD45+Lin?CRTH2+ lymphoid cells (Determine 1A) and were detected at a mean frequency of 0.15% of CD45+ cells in the blood, 0.04% in the lungs and 0.06% in lung lymph nodes (Figure 1B). Human ILC2s highly express both the integrin chains for 2 integrins (CD18 and CD11a) and moderately express both the integrin chains for 1 integrins (CD29 and CD49d) (Figures 1CCE and Supplement Figures E1CE2). Compared with human CD4 cells, human ILC2s express comparable levels of 1 and 2 integrin adhesion molecules (Physique 1FCH). Open in a separate window Physique 1 Human ILC2s express CD18/CD11a (2/L) and CD29/CD49d (1/4) integrins(A) Representative ILC2 gating strategy from human lymph nodes. (B) The frequency of ILC2s from CD45+ human PBMCs, lungs, and lung lymph nodes. Summary of ILC2 adhesion molecule expression from (C) PBMCs (n=7), (D) lung tissue (n=8), and (E) lymph nodes (n=7). Comparison of ILC2 and CD4+CRTH2+ cell adhesion molecule gMFI from (F) PBMCs (n=5), (G) lungs (n=3), and (H) lymph nodes (n=5) of the same donor. represent ILC2 populations and represent CD4+CRTH2+ populations. Data depicted as boxplots (whiskers: 10thC90th percentile) or individual points; mean represented as a line SEM. *test. Mouse ILC2s express 1 and 2 integrins To study the functional importance of ILC2 expression of adhesion molecules to recruitment of ILC2 to the lung, we used a well validated model of allergen challenge in non-sensitized wildtype mice which induces a rapid innate mediated increase in the number of ILC2s.13 To validate that mouse ILC2s expressed a similar profile of adhesion molecules as.