Th9 cells, a subset of T helper cells producing the cytokine IL-9, were first characterized in mice five years ago. In a study recently published in Science Translational Medicine, Schlapbach et al. investigate the relevance of Th9 cells in humans and their potential contribution to immune responses in the skin.
Helper T cells (CD4+ T cells) play an important role in the orchestration of adaptive immune responses by secreting lineage-specific cytokines and steering the immune response to best address the threat at hand. Several helper T cell (Th) subsets have been described, each usually defined by a signature cytokine and/or transcription factor: Th1 (IFNγ, T-bet), Th2 (IL-4, Gata3), Th17 (IL-17, RORγt), follicular T helper cells (IL-21, Bcl6), and regulatory T cells (Foxp3). It is however important to keep in mind that these seemingly clear-cut lineages are only so for the sake of having a rapid way of defining them, and that there is actually considerable flexibility and plasticity among the Th subsets.
In 2008, two research groups independently reported the identification of another Th subset characterized by the production of the cytokine IL-9 (here and here). Dubbed Th9 cells, these cells could be generated in vitro by activating mouse naïve T cells in the presence of TGFβ and IL-4. Studies using murine disease models subsequently showed that Th9 cells could play a protective role (immune responses against tumors or parasites) as well as a pathogenic one (allergy, colitis, experimental autoimmune encephalomyelitis).
So far, not much is known about human Th9 cells. They can be generated in vitro under conditions similar to those used to induce murine Th9 cells, and they have also been isolated ex vivo. A few studies have looked at the numbers of Th9 cells in blood from allergic patients and in biopsies from melanoma patients, but otherwise most studies on human Th9 cells have focused on in vitro work. In their article recently published in Science Translational Medicine, Schlapbach et al. tried to address the in vivo relevance of Th9 cells in humans, investigating their prevalence, characteristics and function.
- STUDY RESULTS
In a first set of experiments, the authors isolated memory T cells from human peripheral blood, separated them in either skin-tropic T cells (CLA+), gut-tropic T cells (α4β7+), or other (CLA- α4β7-), and stimulated them in vitro with monocytes pulsed with different pathogens commonly found within the skin, the gut, or the lungs. Substantial IL-9 production was only observed in skin-homing effector/memory T cell cultures stimulated with Candida albicans. By contrast, cytokines associated with other major Th cell subsets (IFNγ for Th1, IL-13 for Th2 and IL-17 for Th17) were produced by all tissue-homing memory T cells – at variable levels depending on the tissue-homing subset and the pathogen used for stimulation).
To make sure that the observed restriction of IL-9 production to skin-homing T cells was not due to the limited choice of pathogens used for stimulation, the researchers also polyclonally stimulated the different tissue-homing memory T cells (with a combination of anti-CD3, anti-CD28 and anti-CD2 antibodies). Again, IL-9 production was essentially observed in skin-tropic CLA+ T cell cultures, although in this case some IL-9 production was also detected in other tissue-homing T cell cultures. Whether this means that Th9 cells are also present within the gut-homing and lung-homing memory T cell populations, albeit at lower frequency than in the skin-homing population, or whether the IL-9 production observed is due to other Th subsets also being capable of producing IL-9 remains unclear.
A kinetic analysis of cytokine production by polyclonally-activated skin-homing memory T cells showed that IL-9 was transiently expressed, with production peaking early at day 2 after stimulation and levels decreasing rapidly by day 4. By contrast, the levels of other Th cytokines (IFNγ, IL-13 and IL-17) increased less rapidly but more constantly after stimulation, up until day 8. Two days after stimulation, when IL-9 levels were highest, flow cytometry analysis of IL-9-producing CLA+ effector/memory T cells showed that very few IL-9+ cells co-expressed IFNγ, IL-13 or IL-17, suggesting that IL-9 is produced by a distinct Th9 cell population, at least within this time window after stimulation.
In another set of experiments, Schlapbach et al. isolated resident memory T cells from human skin, small intestine, and lung, and polyclonally activated them. IL-9 production was only observed in cultures of skin-resident T cells. Since human innate lymphoid cells (ILCs) have also been reported to produce IL-9 upon stimulation with IL-33 and IL-2, the authors checked whether the IL-9 they detected in the skin cell cultures could come from ILCs, however they could not detect any IL-9 after stimulating the cultures with IL-33 and IL-2. Flow cytometry analysis further confirmed that CD3+ T cells were the major source of IL-9 in skin cell cultures, both before and after polyclonal T cell activation.
As observed in the case of circulating CLA+ T cells, IL-9 production by skin-resident Th cells was transient. Although the majority of IL-9-producing skin-resident T cells did not co-express IFNγ, IL-13 or IL-17 four days after stimulation, up to 20-30% did. The authors looked at the production of other cytokines, and found that IL-9-producing skin resident Th cells did not co-express IL-10, IL-22 or Foxp3 either, but did co-express the pro-inflammatory cytokine TNFα and the cytotoxic molecule granzyme B. It is interesting to note that human IL-9-producing T cells differ from mouse Th9 cells in that they do not produce IL-10.
Since IL-9 levels peaked earlier than the levels of other Th cytokines following memory T cell stimulation, Schlapbach et al. hypothesized that IL-9 might contribute to cell activation and cytokine production. They therefore isolated CLA+ T cells from blood and T cells from skin and stimulated them in presence or absence of an anti-IL-9 blocking antibody. They found that two days after stimulation, the levels of IL-9, IFNγ, IL-13 and IL-17 produced by the polyclonally-activated T cells were lower in the presence of IL-9-blocking antibodies. However, there is no data for later time points, so whether blocking IL-9 has any long-term effect or not is unknown. Considering the rapid and transient production of IL-9, however, these data suggest that IL-9 is at least important for early T cell cytokine production (both IL-9 and other cytokines).
Finally, Schlapbach et al. complete their study by looking at the potential relevance of IL-9 and Th9 cells in human skin diseases. Using immunohistochemistry, they show that IL-9-producing cells are present in skin lesions of psoriatic patients in higher numbers than in healthy skin. Similarly, the numbers of IL-9+ cells in atopic dermatitis skin lesions tend to be higher than in healthy skin, however the difference is not statistically significant. Using immunofluorescence, the authors further show that part of the IL-9+ cells detected in skin are CD3+CD4+ Th cells, but they do not however investigate what the other IL-9-producing cell types present might be.
- SUMMARY
To sum up, the study shows that human Th9 cells are essentially found within the pool of skin-homing effector/memory T cells in the circulation, and are also present in skin, but not in lung or small intestine. Interestingly, a sizeable proportion of circulating skin-tropic Th9 cells were specific for Candida albicans, suggesting that Th9 cells may play a role in the protection against extracellular pathogens. Based on their findings, the authors suggest that Th9 cells may initiate and/or amplify inflammation in skin by producing TNFα and/or enhancing activation and cytokine production by Th1, Th2, Th17 and Th9 cells. This hypothesis is further supported by the observed increased numbers of IL-9-producing cells in psoriatic skin lesions compared to healthy tissue.
Importantly, IL-9 production by both circulating and skin-resident T cells appears transient, similar to what is seen for murine in vitro-differentiated Th9 cells. The fact that at peak IL-9 production IL-9+ T cells mostly do not co-express any other major Th cytokine suggests that IL-9-producing T cells are a discrete population. However the long-term stability of such Th9 cells is unclear. Further studies are needed to determine whether human Th9 cells differentiate into other Th subsets or whether they remain a distinct Th subset, albeit one that may no longer express IL-9.
References
1. Human TH9 Cells Are Skin-Tropic and Have Autocrine and Paracrine Proinflammatory Capacity. Schlapbach C, Gehad A, Yang C, Watanabe R, Guenova E, Teague JE, Campbell L, Yawalkar N, Kupper TS, Clark RA. Sci Transl Med. 2014 Jan 15;6(219):219ra8. doi: 10.1126/scitranslmed.3007828
PMID: 24431112
2. IL-4 inhibits TGF-beta-induced Foxp3+ T cells and, together with TGF-beta, generates IL-9+ IL-10+ Foxp3(-) effector T cells. Dardalhon V, Awasthi A, Kwon H, Galileos G, Gao W, Sobel RA, Mitsdoerffer M, Strom TB, Elyaman W, Ho IC, Khoury S, Oukka M, Kuchroo VK. Nat Immunol. 2008 Dec;9(12):1347-55. doi: 10.1038/ni.1677
PMID: 18997793
Free access in PMC.
3. Transforming growth factor-beta ‘reprograms’ the differentiation of T helper 2 cells and promotes an interleukin 9-producing subset. Veldhoen M, Uyttenhove C, van Snick J, Helmby H, Westendorf A, Buer J, Martin B, Wilhelm C, Stockinger B. Nat Immunol. 2008 Dec;9(12):1341-6. doi: 10.1038/ni.1659
PMID: 18931678
Schlapbach C, Gehad A, Yang C, Watanabe R, Guenova E, Teague JE, Campbell L, Yawalkar N, Kupper TS, & Clark RA (2014). Human TH9 Cells Are Skin-Tropic and Have Autocrine and Paracrine Proinflammatory Capacity. Science translational medicine, 6 (219) PMID: 24431112
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