Several studies demonstrate the existence in experimental animal models of a novel subset of Th effectors that are distinct from the classic Th1 and Th2, and that have been named Th17 because of their ability to produce IL-17. These cells represent a distinct lineage that originates mainly in the presence of TGF-β and IL-6 and need the presence of IL-23 for their expansion and/or maintenance. IL-23 is a member of the IL-12 cytokine family, which shares the p40 subunit with IL-12 and differs in the expression of p19 instead of p35. At least in mice, Th17 cells arise as a part of mucosal host defense and their major role seems to be protection against infections sustained by extracellular bacteria (33
), but under certain circumstances they can also be involved in the pathogenesis of chronic inflammatory disorders, including some models of autoimmune diseases (9
). Notably, in these models, IFN-γ produced by Th1 cells, which are crucial for the protection against intracellular bacteria, does not appear to be pathogenic, but instead appears protective, as inhibition of IFN-γ signaling enhances the development of pathogenic Th17 and exacerbates autoimmunity (20
). Even the neutralization of IL-4 produced by Th2 is critical in neutralizing the development of IL-17; however, neither IFN-γ nor IL-4 seem to be effective on already established Th17 (20
Currently, very little is known about human Th17. Some studies have shown that the presence of IL-17 mRNA or IL-17 protein in tissues or biological fluids of subjects with different autoimmune disorders (23
) or other chronic inflammatory diseases (28
). The results of this study provide evidence of the existence of increased numbers of CD4+ T cells producing IL-17 in the disease-affected gut areas of subjects with CD, compared with either PB or apparently healthy gut areas. Based on this finding, IL-17–producing CD4+ T cells derived from the gut of CD subjects were expanded in vitro and cloned to get an amount of these cells suitable for phenotypic and functional investigations.
The first information produced by our study was that a remarkable proportion of IL-17–producing CD4+ T cells share the ability to produce IFN-γ, whereas CD4+ T cells producing both IL-17 and -4 were never observed. The cells producing both IL-17 and IFN-γ, which we named Th17/Th1, were not the result of an in vitro artifact, as about one third of ex vivo–derived IL-17–producing CD4+ T cells already showed a double IL-17+IFN-γ+ phenotype. When examined at clonal level, Th17 and Th17/Th1 shared different phenotypic properties from the other types of clones, some of which appearing to be selective. For example, only Th17 and Th17/Th1 clones exhibited the expression of IL-23R. Interestingly, the IL-23R did not apparently play any role in the expansion of these cell subsets, because they did not proliferate in response to IL-23 alone, and IL-23 did not potentiate their anti-CD3/CD28 Ab–induced proliferation. Thus, it is probable that IL-23 is not important for human Th17 expansion, but rather for their survival and/or maintenance. In contrast, under the same conditions, anti-CD3/28–stimulated Th17 and Th17/Th1 clones proliferated in response to IL-2, -12, or -15. The ability of IL-2 to promote the proliferation of human Th17, a finding that was also supported by the observation that IL-2 was the growth factor used in this study to expand Th17 from both PB or gut mucosa and to get Th17 clones, is apparently at variance with recent results showing that IL-2 constrains generation of mouse Th17 cells (36
). The reason for such a discrepancy is presently not clear. Among chemokine R, both Th17 and Th17/Th1 clones apparently lacked CXCR3-A, CXCR3-B, CCR3, CCR8, and CCR9, but exhibited high levels of CXCR4 and CXCR6. Th17 clones also expressed significantly higher levels of CCR4 and CCR5 than the other types of clones, and both Th17 and Th17/Th1 selectively expressed CCR6. CCR4 expression has been associated with the ability of cells to traffic into peripheral tissues (37
). CCR6 has been found to be expressed by B cells, DCs, and memory, but not naive, T cells. However, its ligand, MIP-3α/CCL20 does not exert chemotactic activity on B cells, but only on memory T cells (38
). Interestingly, however, CCR6 expression by memory T cells is lost after their prolonged TCR triggering (39
). Thus, the selective expression of CCR6 by Th17 and Th17/Th1, but not by Th1, Th2, or Th0, clones may mean that Th17 and Th17/Th1 are the only memory T cells that continue to express CCR6 even after prolonged antigen activation, thereby maintaining the possibility of recruitment in response to MIP-3α/CCL20. This finding may have important implications for the long-term maintenance of Th17 influx, supporting their important pathogenic role into the inflamed tissues.
Human Th17 and Th17/Th1 clones also shared some peculiar functional properties. We found that both types of clones were able, as Th1 clones, to provide B cell help for the production of IgM, IgG, and IgA, but not IgE, Ab. In contrast, as Th2 clones, they showed poor granzyme A expression and low cytotoxic capability, thus displaying an intermediate functional pattern in regard to B cell helper and cytotoxic activity between Th1 and Th2 clones. However, the most impressive functional feature of both Th17 and Th17/Th1 clones was their lower susceptibility, in comparison with both Th1 and Th2 clones, to the suppressive activity of an autologous T reg cell clone derived from circulating CD4+CD25highFoxp3+ cells. Although the mechanism for the higher resistance of Th17 and Th17/Th1 to the action of T reg cells is still unclear, this finding can provide additional support for an important role of IL-17–producing cells in the maintenance of the inflammatory processes in autoimmune disorders.
The receptor expression and functional capabilities observed in T cell clones derived from the affected areas of gut from subjects with CD were neither related to their pathological source nor to some in vitro artifact referable to the cloning procedure. Indeed, similar receptor expression and function were found in T cell clones derived from healthy gut areas of subjects who underwent colectomy because of colon carcinoma. More importantly, the existence of Th17 and Th17/Th1 cells, as well as their expression of CCR6, IL-23R, and RORγt was also observed in freshly derived CD4+ T cells from gut, PB, and tonsil. In this respect, it is of note that sorting of CCR6+ cells allowed to obtain populations strongly enriched in IL-17–producing cells, suggesting that this marker may be useful to get high numbers of Th17 cells and, therefore, of potentially great help in clarifying their pathophysiologic role in humans. Of note, during the revision process of this paper, a study was published (40
) that also reports preferential expression of CCR6 and RORγt by human PB Th17 cells.
Another important finding emerging from this study was the apparent discrepancy with some results reported in mice in regard to the relationship between Th17 and Th1. In a first proposed mouse model, it was suggested that the early differentiation of Th1 and Th17 from naive CD4+ T cell precursors was shared, and thus Th1 and Th17 diverged contingent upon the selective availability of IL-12 or -23 acting on a common “Th1 precursor” or “pre-Th1” intermediate that coexpressed IL-12R and -23R (41
). However, in a subsequently proposed mouse model, which was based on the demonstration that the differentiation into Th17 depends upon TGF-β and IL-6 rather than upon IL-23, it was suggested that Th1 and Th17 subsets were not overlapping and represented distinct lineages (19
). In this study, we do not provide any evidence for the mechanisms responsible for the differentiation of human Th17, as well as for the question of whether in humans, as in mice, Th17 and Th1 represent distinct lineages. However, we not only demonstrated the ex vivo existence of CD4+ T cells able to produce both IL-17 and IFN-γ but we were also able to induce Th17 clones to produce IFN-γ in addition to IL-17 after their culturing in the presence of IL-12. The possible relationship between human Th17 and Th1 was also supported by the demonstration that Th17 clones expressed not only RORγt, a finding that is in agreement with the results reported in mice (21
), but also remarkable amounts of the Th1 transcription factor T-bet, and that amounts of both T-bet and RORγt were comparable in human Th17 and Th17/Th1 clones.
Interestingly, the ability of IL-12 to induce Th17 cells to produce IFN-γ, in addition to IL-17 associated with the up-regulation of T-bet and the down-regulation of both RORγt expression and IL-17 production in Th17 clones. This means that in humans, even established Th17 clones, independent of whether they have a distinct or common origin with Th1, are responsive to IL-12 and are still sufficiently flexible to acquire the ability to produce IFN-γ. These findings strongly suggest that in human Th17 both RORγt and T-bet can play an important modulatory role. The possibility that T-bet–dependent signaling was responsible for the increased production of IFN-γ by our T cell clones was not directly explored in this study, and thus remains unproved. However, it has recently been shown that ectopic T-bet expression in mouse Th17 cells can promote IFN-γ secretion and decrease IL-17 production, suggesting that even in mice the Th17 phenotype is not stable and can give rise to IFN-γ production in vitro via T-bet–mediated signaling (42
). In this respect, it should be taken into account that other recent data in mice, again, raise the question of the developmental relationship between Th1 and Th17. First, the existence of a dual population of IL-17- and IFN-γ–producing cells has also been reported in studies performed in mice (42
). Second, T-bet has been found to be required for optimal IL-17 production in the presence of IL-23 (44
). Finally, therapeutic administration of a small interfering RNA specific for T-bet significantly improved the clinical course of established EAE by limiting the differentiation of autoreactive Th1 and inhibiting pathogenic Th17 via regulation of IL-23R (45
). These findings suggest that even in mouse models T-bet and RORγt may play distinct, yet complementary, roles in the development of Th17 cells. In this study, the IL-12–induced IFN-γ production by Th17 clones was partially inhibited by the contemporaneous presence of IL-23. Because a similar inhibitory effect by IL-23 on the IL-12–induced IFN-γ production could also be observed in Th1 clones, which do not express IL-23R, it is reasonable to suggest that it was mediated by the ability of IL-23 to bind both IL-23R and IL-12Rβ1 via the p40 chain shared between IL-12 and -23, which therefore display binding competition. Because IFN-γ is a well known inducer of IL-12Rβ2 (46
), it can strongly influence the susceptibility of Th17 to the activity of IL-12, thus favoring the shifting of these cells toward the Th1 phenotype. This finding may also account for the reported inhibitory activity of IFN-γ on the development of mouse Th17, as well as for its protective effects (47
In conclusion, our data provide the first detailed phenotypic and functional characterization of human Th17 isolated from the disease-affected gut mucosa of subjects with CD, as well as from normal tissues, showing that Th17 display distinct functional properties from Th1 or Th2 cells and identifying IL-23R, CCR6, and RORγt as Th17–specific markers. In addition, we describe a new subset of IFN-γ–producing Th17 sharing features with both Th1 and Th17, which has not been previously reported in mice. This novel subset exists in vivo in humans and can be induced in vitro by stimulating Th17 in presence of IL-12, thus raising new issues on the Th17 developmental and/or functional relationship with Th1.