It is important to call attention to the fact that CCL25 induced the migration of IL-17+ cells without affecting IL-17 production in vitro. Therefore, the modulation of pleural IL-17 levels by CCL25 is actually a result of the in vivo migration of IL-17+ γδ T lymphocytes. IL-17+ γδ T lymphocytes are committed as IL-17 producers within the fetal thymus, via RORγt transcriptional factor- and Notch-Hes1-dependent mechanisms, independently of TCR signaling [[43-46]] and CD27 costimulation [[47]]. In the immune site, their activation is determined by multiple cytokines, among which a chief role is attributed to IL-1β and IL-23 in mice and humans [[48-50]]. It has been shown that TCR γδ+/CCR6+ cells that

produce IL-17 coexpress CCR9 (which was not observed for γδ+/NK1.1+ cells, which produce IFN-γ) [[6, FGFR inhibitor Ku-0059436 clinical trial 34]]. Accordingly, CCL25 induces the specific chemotaxis of Th17 CD4+ T cells polarized by retinoic acid (which is an important regulatory signal in the intestine), but not of Th0 lymphocytes [[51]]. These T cells were shown to express CCR9/α4β7 integrin and preferentially migrate to the intestine and regulate inflammation. Moreover, it has been demonstrated that, throughout Th17 differentiation, CD4+ T lymphocytes are shown to increase the expression of chemokine receptors, including CCR9 [[52]]. Interestingly, IL-17-producing γδ T cells

have been shown to be CD25+ and CD122− [[43, 44]], a phenotype observed by us on γδ T cells recovered from CCL25-stimulated mouse pleura. It is noteworthy that, since CCL25 i.pl. injection failed to trigger CD122+ T-cell migration, the percentage of CD122+ γδ T lymphocyte population in the pleura of CCL25-stimulated mice slightly decreased (SAL 11.8% versus CCL25 5.0% among γδ T lymphocytes). Similar to our data, it has been demonstrated that IL-17-producing γδ T cells did not produce IFN-γ or IL-4

and specifically expressed CD25 but not CD122 (whereas CD122+ γδ T lymphocytes produced IFN-γ). Moreover, IL-17-producing γδ T lymphocyte maintenance was shown to depend on CD25 and IL-2 [[44]]. It has been also shown that CD122lo γδ T cells recovered from mouse spleen, lymph nodes, and thymus produced high levels of IL-17 but small amounts or no IFN-γ upon Idoxuridine TCR in vitro stimulation [[43]]. An inverse correlation between CD122 and CCR9 has also been demonstrated on γδ lymphocytes from mouse thymus [[53]]. This work demonstrates that γδ thymocytes that express high levels of CCR9 are CD122lo, whereas CCR9lo express high levels of CD122. It is important to note that the CCL25 neutralization and α4β7 integrin blockade during allergic pleurisy did not inhibit αβ T lymphocyte recruitment, whereas the i.pl. stimulus with CCL25 selectively triggered γδ T-cell migration. These data corroborate and reinforce the hypothesis that CCL25 is important for the migration of a specific γδ T-cell subset that produces IL-17 during an allergic reaction, via α4β7 integrin.