PPAR-γ regulates the effector function of human T helper 9 cells by promoting glycolysis – Nature Communications

In vitro and in vivo primed T_H9 cells display key features of pathogenic T_H2 cells

The transcriptomic signature of pT_H2 cells has been previously identified by single-cell analysis of T cells extracted from multiple T_H2-driven diseases². To test whether human in vitro primed T_H9 cells recapitulate the core pT_H2 cell phenotype, we differentiated naive T cells into T_H1 (IL-12), T_H2 (IL-4), T_H9 (IL-4+TGF-β), and iT_REG (TGF-β) cells and performed transcriptomic profiling using RNA sequencing (RNA-seq) at day 7. Pairwise comparison to other subsets showed that 1492 genes were specifically upregulated in T_H9 cells (Fig. 1a). We then compared our T_H9 transcriptome with three pT_H2-specific transcriptomes identified in EoE⁴, allergic asthma⁵, and allergen-specific T_H2 cells⁷, respectively (Fig. 1b). PPARG, IL5, IL17RB, and IL9R, which are hallmarks of pT_H2 cells, were upregulated in T_H9 cells as well as in all three pT_H2 datasets, while IL9 was upregulated in two of the three (Fig. 1c and Supplementary Fig. 1a). In contrast, SPI1, encoding the transcription factor PU.1, previously shown to be associated with IL-9 expression^16,17, was neither expressed in pT_H2-specific transcriptomes, nor in T_H9 cells (Supplementary Fig. 1a). High levels of PPAR-γ were confirmed at the protein level in both in vitro primed T_H9 cells and in vivo primed T_H9 clones, which we generated from ex vivo isolated memory T_H cells that were sorted based on chemokine receptor expression (Fig. 1d–f). In summary, these findings strongly support our hypothesis that in vitro and in vivo primed T_H9 cells share key similarities with pT_H2 cells.

As we had previously identified PPAR-γ⁺ T_H9 cells in ACD⁹, we next considered validating the association between the T_H9 and pT_H2 phenotypes in acute allergic skin inflammation. Time course transcriptomics of untreated non-lesional (NL) skin and positive patch test reactions of lesional skin to nickel at 24 h, 48 h, and 120 h post allergen application showed a marked upregulation of the pT_H2-associated genes in ACD (Fig. 1g and Supplementary Fig. 1b). Across individual samples, the expression of IL9 correlated with the expression of IL13 (R² = 0.80; P < 0.0001), IL5 (R² = 0.54; P < 0.0001), IL31 and IL19, but not IFNG (Fig. 1h and Supplementary Fig. 1c), consistent with the predominance of T_H9 cells in the T_H2 cell pool. Due to the fact that PPARG is expressed in various skin cell types, including keratinocytes⁹, the correlation analysis of PPARG with IL9 does not allow any conclusion with regard to T_H9 cells.

Collectively, these data show that both in vitro and in vivo primed T_H9 cells express the core features of pT_H2 cells, including upregulated expression of PPAR-γ, IL-5, IL-9, and IL-9R. They can thus serve as model cells for studying the functional role of PPAR-γ in human T_H cells. Further, human ACD appears to be a valid model for studying the functionality of T_H9 cells ex vivo.

PPAR-γ mediates the high glycolytic activity of T_H9 cells

To investigate the role of PPAR-γ in human T_H cells, we first assessed the transcriptional response of activated T_H9 clones to treatment with GW9662, a potent PPAR-γ antagonist. Pathway analysis of RNA-seq data revealed concerted downregulation of genes associated with T cell activation, glucose metabolism, and aerobic glycolysis (Supplementary Fig. 2a). At the single gene level, mRNA expression of all aerobic glycolysis enzymes was significantly downregulated by PPAR-γ inhibition (Fig. 2a, b). This prompted us to further analyze the role of PPAR-γ in aerobic glycolysis of T_H9 cells. In vitro primed T_H9 cells showed higher glycolytic activity than T_H1- or T_H2-primed T cells after activation with αCD3/CD2/CD28 (Fig. 2c and Supplementary Fig. 2b). To verify whether PPAR-γ was involved in this process, we starved in vitro primed T_H9 cells in glucose-free medium in presence or absence of GW9662. We then performed measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in real-time before and after activation with αCD3/CD2/CD28 in either low or high-glucose environments. PPAR-γ inhibition by GW9662 or the alternative PPAR-γ antagonist, T0070907, hampered the glycolytic response in high- but not low-glucose environments, particularly following T cell activation (Fig. 2d, e and Supplementary Fig. 2c, d). These findings were corroborated by measurements of glucose uptake, in which in vitro primed T_H9 cells showed higher glucose uptake compared to T_H1, and T_H2 cells (Fig. 2f). Importantly, glucose uptake in T_H9 cells was reduced by PPAR-γ-inhibition or by siRNA-induced PPARG knockdown (Fig. 2g).

a Intermediates and enzymes (red) of aerobic glycolysis. b RNA-seq of T_H9 clones incubated in presence of GW9662 for 48 h and activated by αCD3/CD2/CD28 for 12 h. c Maximal glycolytic capacity of in vitro primed T_H cells in the resting state and 24 h after activation with αCD3/CD2/CD28. d ECAR measurements of in vitro primed T_H9 cells cultured in media with glucose of different levels and GW9662 for 48 h and activated by injection of glucose and αCD3/CD2/CD28. e Maximal glycolytic capacity of in vitro primed T_H9 cells from d. f Glucose uptake by in vitro primed T_H cells measured with fluorescent 2-NBDG uptake by flow cytometry at day 7. g Glucose uptake by naive T_H cells primed under T_H9 conditions for 7 days in presence of GW9662 or transfected with PPARG and control siRNA, respectively. Efficiency of knockdown was determined by measuring PPARG levels after transfection by RT-qPCR (right). h Glucose uptake of in vivo primed effector memory T_H cells (T_EM) sorted by flow cytometry into T_H1, T_H2, and T_H9 cells according to their chemokine receptor profile. Sorted T_H cells were incubated in presence or absence of GW9662 for 48 h, and activated by αCD3/CD2/CD28 for 4 h. i Proliferation of in vitro primed T_H9 cells, activated by αCD3/CD2/CD28 for 4 days in presence or absence of GW9662 and glucose of different levels, measured with CFSE dilution by flow cytometry. The data are representative of one experiment with three clones from one donor (b) or independent experiments with three (d, g (left), i), four (c, f (T_H1), h), five (e, f (T_H2 and T_H9), g (right)) or six (g, right) donors. Statistics: b differences between treatment groups were calculated as an adjusted log-fold change, and hypothesis testing was performed using the Benjamini–Hochberg adjusted p value (DESeq2). c, f, h One-way ANOVA, followed by a Tukey’s test for multiple comparisons. g Two-tailed paired t test. e, i One-way ANOVA, followed by a Šidák’s test for multiple comparisons. The data are presented as mean ± SD. Only p values <0.05 are shown.

We next expanded our findings to in vivo primed memory T_H cells, leveraging the ability to sort different subsets ex vivo based on their chemokine receptor profile, including CXCR3^–/CCR4⁺/CCR6^–/CCR8⁺ T_H9 cells^9,18. Consistent with our previous observations, in vivo primed T_H9 cells showed higher glucose uptake compared to other T_H cell subsets, and GW9662 significantly inhibited the glycolytic activity in T_H9 cells but not in T_H2 or T_H1 cells (Fig. 2h).

Given the central role of aerobic glycolysis for T cell proliferation¹⁹, we finally tested whether PPAR-γ inhibition affected TCR stimulation-induced proliferation in low and high-glucose environments. Both GW9662 and T0070907 significantly reduced αCD3/CD2/CD28-induced proliferation in high-glucose environments (Fig. 2i and Supplementary Fig. 2e, f).

Since PPAR-γ has been implicated in mediating fatty acid (FA) uptake in activated T_H cells^20,21, we examined FA metabolism in response to PPAR-γ antagonism. In vitro and in vivo primed T_H9 cells did not exhibit higher FA uptake compared with T_H1 and T_H2 cells (Supplementary Fig. 2g) and PPAR-γ antagonism had no effect on FA uptake (Supplementary Fig. 2h, i). In addition, glutamine uptake of T_H9 was not affected by PPAR-γ inhibition, suggesting that glutaminolysis is not primarily regulated by PPAR-γ in these cells (Supplementary Fig. 2j).

PPAR-γ has previously been shown to regulate the expression of IL-33R in murine T_H2 cells, thereby increasing their sensitivity to the tissue alarmin IL-33^11,12 in allergic inflammation. While IL-33R (IL1RL1) is also associated with the pT_H2 phenotype in humans^4,6,7,8,22, PPAR-γ antagonism did not downregulate its expression in T_H9 cells (Supplementary Fig. 2k).

Collectively, these data strongly suggest that both in vitro and in vivo primed human T_H9 cells are characterized by high glycolytic capacity post-TCR-stimulation. Moreover, in the setting studied here, PPAR-γ signaling appears to be a crucial mediator of glycolysis, whereas FA oxidation and glutaminolysis remain unaffected.

High glycolytic activity in T_H9 cells differentially regulates cytokine expression

PPAR-γ is required for the full effector function in T_H9 and pT_H2 cells, including the production of proinflammatory cytokines^9,11,12. Based on our findings, we hypothesized that PPAR-γ might control cytokine production indirectly, namely by promoting glycolysis. To test this, we cultured in vitro primed T_H9 cells in media containing different glucose concentrations and measured their cytokine profiles at day 7. Production of the pT_H2 marker cytokine IL-9 showed a strong dependency on glucose availability, whereas production of IL-13, the conventional T_H2 cytokine, did not (Fig. 3a). Further, in vivo primed T_H9 clones cultured in different glucose concentrations downregulated the production of IL-9 but not IL-13 in low-glucose environments (Fig. 3b).

a Cytokine expression measured by flow cytometry of T_H9 cells primed in vitro in media containing glucose of different levels for 7 days. b Cytokine expression of in vivo primed T_H9 clones cultured for 72 h in media containing glucose of different levels, measured as in a. c In vitro primed T_H9 cells were activated for 4 h with αCD3/CD2/CD28, then sorted by flow cytometry based on their glucose uptake measured by 2-NBDG uptake (left). Cytokine expression in the sorted T_H cell populations was measured by RT-qPCR (right). d Cytokine expression of in vivo primed T_H9 cells cultured for 7 days in the presence of 2-DG, measured as in a. e Cytokine expression of in vitro primed T_H9 cells cultured for 48 h in media containing glucose of different levels and in presence or absence of GW9662, measured as in a. The data are representative of independent experiments with three (e), six (c), or seven (a) donors or fourteen clones from two donors (b, d). Statistics: a, c One-way ANOVA, followed by a Tukey’s test for multiple comparisons. b, e One-way ANOVA, followed by a Šidák’s test for multiple comparisons. d Two-tailed paired t test. The data are presented as mean ± SD. Only p values <0.05 are shown.

To demonstrate a direct relationship between high-glucose metabolism and cytokine production, we next sorted in vitro primed T_H9 cells according to their glucose uptake level, measured by the uptake of 2-NBDG. Subsequently, we performed RT-qPCR for IL9, IL13, and PPARG. Glucose uptake correlated with the expression of IL9 and PPARG, but not IL13 (Fig. 3c and Supplementary Fig. 3c). Taken together, these data strongly suggests that glycolytic activity regulates the expression of IL-9. Similar regulation, albeit less pronounced, was observed for IL5 expression (Supplementary Fig. 3a–c).

In a next step, we hence inhibited glycolysis in T_H9 cells using the glucose analog 2-deoxy-d-glucose (2-DG) and the aerobic glycolysis inhibitor lonidamine (LND) to investigate the effect on cytokine expression. In T_H9 cells primed in vivo, 2-DG and LND inhibited the expression of IL-9 and IL-5 but not IL-13 (Fig. 3d and Supplementary Fig. 3d, e). Finally, PPAR-γ antagonism in high-glucose environments reduced the production of IL-9 to the levels seen in low-glucose environments, whereas IL-13 levels remained unaffected neither by PPAR-γ antagonism nor by low-glucose availability (Fig. 3e).

To investigate the contribution of FA metabolism to the regulation of cytokines in T_H9 cells, we analyzed IL-9 expression in response to FA inhibition. Neither did culturing of T_H9 clones in FA-free medium affect IL-9 or IL-13 expression (Supplementary Fig. 3f), nor did inhibition of FA metabolism with etomoxir, an inhibitor of carnitine palmitoyltransferase-1 (Supplementary Fig. 3g).

Taken together, these observations indicate a dichotomous role of glycolytic activity in regulating the production of IL-9, IL-5, and IL-13 by activated T_H9 cells.

mTORC1 integrates glycolytic activity with the effector function in T_H9 cells

We next investigated the mechanisms underlying the association between glycolysis and cytokine production in activated T_H9 cells. Mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of cellular metabolism and effector functions in T cells. Nutrients, such as glucose, are critical activators of mTORC1²³. Furthermore, the mTORC1-hypoxia-inducible factor-1α (HIF-1α) pathway is necessary for the expression of IL-9 in murine T cells, with HIF-1α binding directly to the Il9 promoter and activating its transcription^24,25,26. Given the role of the established mTORC1-HIF-1α-IL-9 axis and our previous results, we hypothesized that mTORC1 might mediate the PPAR-γ-dependent expression of IL-9 in T_H9 cells.

Phosphorylation of mTORC1 in activated T_H9 cells measured by phosphorylated S6 (pS6) was glucose-dependent and reduced by PPAR-γ inhibition under high-glucose conditions (Fig. 4a, and Supplementary Fig. 4a, b). Indeed, IL-9⁺ T cells were strongly enriched in the pS6⁺ cell population, whereas the proportion of IL-13⁺ T cells was similar in pS6^– and pS6⁺ populations (Fig. 4b, c). Moreover, inhibition of mTORC1 by either siRNA against RPTOR or by rapamycin decreased the production of IL-9 but not IL-13 in activated T_H9 cells (Fig. 4d, e and Supplementary Fig. 4c, d).

a Western blot analysis of pS6 in T_H9 cells primed in vitro. b In vitro primed T_H9 cells were cultured in glucose of different levels for 48 h, and pS6 and IL-9 were measured 18 h after activation with αCD3/CD2/CD28 by flow cytometry. The histogram shows pS6 positive cells, split into high and low pS6. Dot-plots represent IL-9⁺/IL-13⁺ clusters. c The IL-9⁺/IL-13⁺ ratio in T_H9 cells primed in vitro from b. d, e Cytokine expression of in vitro primed T_H9 cells after incubation in glucose of different levels and rapamycin for 48 h, measured as in b. f Immunofluorescence staining for CD4 and pS6 on skin samples of allergic contact dermatitis (ACD) and quantification of CD4⁺pS6⁺ cells in normal skin (NS) and ACD skin samples. Scale bars, 50 μM. g Cytokine expression of T cells isolated from ACD skin biopsies incubated with GW9662 for 48 h, measured as in b. h Immunofluorescence staining for pS6 and PPAR-γ on skin samples of ACD and quantification of PPAR-γ⁺pS6⁺ cells in NS and ACD skin samples. Scale bars, 50 μM. The data are representative of independent experiments with one (g), three (c), four (f (ACD), h (ACD)), five (f (NS), h (NS)), six (a), or nine (e) donors. Statistics: a, c, e One-way ANOVA, followed by a Šidák’s test for multiple comparisons. f, h Two-tailed unpaired t test. g Two-tailed paired t test. The data are presented as mean ± SD. Only p values <0.05 are shown.

Since the PPAR-γ agonist troglitazone (TGZ) unexpectedly reduced IL-9 expression in T_H9 cells, we next investigated the mechanism by which this occurs. Interestingly, pS6 levels were reduced in the presence of TGZ (Supplementary Fig. 4e), suggesting that mTORC1 is inhibited. Previous studies have shown that PPAR-γ agonists, such as TGZ, activate AMP-activated protein kinase (AMPK)²⁷. We, therefore, hypothesized that TGZ-mediated AMPK activation negatively regulates mTORC1, which in turn suppresses IL-9 expression. Indeed, Western blot analysis revealed that TGZ leads to phosphorylation of AMPK and mTORC1 inhibition (Supplementary Fig. 4f). In addition, the AMPK activator A-769662 also reduced IL-9, but not IL-13 levels (Supplementary Fig. 4g). Together, this data strongly supports our hypothesis that IL-9 expression is mTORC1-dependent.

To verify whether T_H9 cells expressed activated mTORC1 in human skin inflammation, we performed immunofluorescence staining of normal skin (NS) and ACD skin samples and isolated T cells from such lesions. Double immunofluorescence revealed that CD3⁺ and CD4⁺ T_H cells that express pS6 are significantly enriched in the infiltrate of ACD compared to NS (Fig. 4f and Supplementary Fig. 4h, i). Virtually all IL-9⁺ T_H cells isolated from ACD show S6 phosphorylation, and thus have active mTORC1 signaling (Supplementary Fig. 4j). Incubation with GW9662 ex vivo showed reduced activation of mTORC1 and a significantly inhibited IL-9 production (Fig. 4g). As it is known that ACD skin is infiltrated by a substantial number of PPAR-γ⁺ T_H cells⁹, we finally investigated whether those cells would show the activation of mTORC1. Indeed, PPAR-γ⁺pS6⁺ double-positive lymphocytes were significantly increased in the dermis of ACD compared to NS (Fig. 4h and Supplementary Fig. 4k).

Collectively, these findings strongly suggest that glucose- and PPAR-γ-dependent production of IL-9 in T_H9 cells is regulated via mTORC1 in acute allergic skin inflammation.

Paracrine IL-9 promotes aerobic glycolysis in IL-9R⁺ T_H cells by inducing the lactate transporter MCT1

After revealing the association between PPAR-γ-dependent glycolytic activity and IL-9 production in T_H9 cells, we hypothesized that paracrine IL-9 might regulate glucose metabolism and downstream effector functions in IL-9R⁺ T_H cells. Previous data suggested that IL9R is preferentially expressed in pT_H2 and T_H9 cells (Fig. 1 and ref. ^4,5,7,8), but these findings had to be confirmed at the protein level and in human skin inflammation. Thus, we first confirmed the expression of IL-9R in T_H9 clones primed in vivo (Fig. 5a), circulating CXCR3⁻/CCR4⁺/CCR8⁺ effector memory T_H cells (Fig. 5b), and T_H cells infiltrating lesions of ACD (Fig. 5c, d), showing that pT_H2 and T_H9 cells are important targets of IL-9 in human skin inflammation. Next, we performed RNA-seq of IL-9R⁺ T_H clones and IL-9R⁺ T_H cells isolated from ACD skin biopsies, incubated with or without IL-9. The pathway analysis of the 250 most IL-9-induced genes showed a coordinated induction of genes involved in aerobic glycolysis (Fig. 5e), most prominently SLC16A1 (Fig. 5f). SLC16A1 encodes the monocarboxylate transporter 1 (MCT1), enabling the rapid export of lactate across the plasma membrane and thereby exerting a glycolytic flux-controlling function²⁸. IL-9-induced expression of MCT1 in T_H9 clones was confirmed at the protein level (Fig. 5g). In contrast, inhibition of JAK3, which is central to IL-9R signal transduction²⁹, by ritlecitinib suppressed IL-9-induced upregulation of SLC16A1 (Fig. 5h).

a IL-9R levels of in vivo primed T_H clones analyzed by flow cytometry. b IL-9R levels of PBMCs stained for their chemokine receptor profiles analyzed by flow cytometry. c Immunofluorescence staining for CD3 and IL-9R on ACD skin. Scale bars, 50 μM. d IL-9R levels of T cells isolated from ACD analyzed by flow cytometry. e–f RNA-seq of IL-9R⁺ T_H cells isolated from blood and ACD in presence of IL-9 shows (e) pathway analysis of the 250 most significant IL-9-induced genes and (f) changes in the expression of selected aerobic glycolysis genes. g Western blot analysis of MCT1 expression in IL-9R⁺ T_H clones incubated with IL-9 or IL-2 for 48 h. h SLC16A1 expression measured by RT-qPCR in IL-9R⁺ T_H clones in presence of JAK3 inhibitor (JAK3-i) ritlecitinib and IL-9 for 24 h. i RNA expression of SLC16A1 in in vitro primed T_H cells after 7 days. j, k Time course transcriptomic data⁹ shows RNA expression levels of SLC16A1 in i and correlation between IL9 and SLC16A1 expression in j. l ECAR measurements of in vivo primed IL-9R⁺ T_H clones incubated with IL-9 for 16 h. m IL-9R⁺ T_H clones incubated with the MCT1 inhibitor (MCT1-i) BAY-8002 or transfected with SLC16A1 siRNA. Extracellular (e.c.) lactate was measured with the Lactate-Glo^TM Assay (Promega) after 48 h in presence of IL-9. The data are representative of one experiment with one (c, l) donor or two (a (T_H17)), three (h), five (a (T_H1)) or twenty-two (a (T_H9)) clones from one donor or independent experiments with eight clones from one (g) or two (e, f) donors or nine clones from two donors (m (left)) or two (m (right)), three (i), five (b) six (j, k) or eleven (d) donors. Statistics: a Two-tailed unpaired t test. b, d, g, m (right) Two-tailed paired t test. e Fisher’s one-tailed test. f Differences between treatment groups were calculated as an adjusted log-fold change, and hypothesis testing was performed using the Benjamini–Hochberg adjusted p value (DESeq2). h, I One-way ANOVA, followed by a Dunnett’s test for multiple comparisons. k Simple linear regression. h, j, I, m One-way ANOVA, followed by a Tukey’s test for multiple comparisons. The data are presented as mean ± SD. Only p values <0.05 are shown.

Next, we investigated the expression and regulation of SLC16A1 in different T_H cell populations. In vitro T_H9 differentiation induced higher levels of SLC16A1 than T_H1, T_H2, or iT_REG differentiation (Fig. 5i). Time course transcriptomics of TCR-stimulated T_H clones⁹ revealed a significantly higher expression of SLC16A1 in T_H9 clones than in T_H1 and T_H2 clones (Fig. 5j), as well as a close correlation between the expression of IL9 and SLC16A1 (Fig. 5k). Finally, a Seahorse experiment confirmed that IL-9R⁺ T_H clones stimulated by IL-9 exhibited strongly elevated ECAR, in line with IL-9-dependent induction of active aerobic glycolysis and efficient cellular export of lactate (Fig. 5l). Accordingly, IL-9 increased extracellular lactate levels in cultured IL-9R⁺ T_H clones, and these levels were suppressed by the addition of BAY-8002, a potent MCT1 antagonist (MCT1-i) or by siRNA-mediated SLC16A1 knockdown (Fig. 5m). To link IL-9, glycolysis and MCT1 expression, we next investigated IL-9 levels in T_H9 cells in presence of MCT1 inhibitor. We show that MCT1 inhibition reduces IL-9, but not IL-13 levels (Supplementary Fig. 5a). On the contrary, low-glucose environments, which in turn lead to reduced IL-9 levels, inhibited the induction of SLC16A1 (Supplementary Fig. 5b).

Taken together, these data indicate that paracrine IL-9 promotes aerobic glycolysis in IL-9R⁺ T_H cells by inducing the lactate transporter MCT1, which controls glycolytic flux.

IL-9 promotes T cell proliferation in high-glucose environments

Considering the crucial role of aerobic glycolysis in supporting T cell proliferation³⁰, we next investigated the functional effects of IL-9-induced MCT1 expression on T cell proliferation. IL-9 induced strong proliferative responses in IL-9R⁺ T_H clones (Fig. 6a), and this proliferative boost was reversed by adding BAY-8002, the MCT1 inhibitor (Fig. 6b). Moreover, IL-9-induced proliferation was dependent on available glucose levels (Fig. 6c), which further supports the notion that IL-9 promotes glycolytic flux in IL-9R⁺ T_H cells to facilitate proliferation.

a Proliferation of in vivo primed IL-9R⁺ T_H clones in presence of IL-9 and different concentrations of IL-2, measured with CFSE dilution by flow cytometry after 3 days. b Proliferation of in vivo primed IL-9R⁺ T_H clones in presence of IL-9 and MCT1 inhibitor (MCT1-i) BAY-8002 measured as in a. c In vivo primed IL-9R⁺ T_H clones were cultured in media containing glucose of different levels for 7 days. Proliferation was measured in the presence of IL-9 with CFSE dilution by flow cytometry as in a. d Glucose concentrations measured with the Glucose-Glo^TM Assay (Promega) of interstitial fluids of lesional skin of positive patch test reactions to different allergens (Supplementary Table S3) 48 h post allergen application and adjacent non-lesional skin biopsies. e Schematic presentation of the main conclusions. The mTORC1-HIF-1α-IL-9 axis has previously been established by others^24,25,26. The data are representative of one experiment with four a or five c clones from one donor or independent experiments with eight clones from one donor b or independent experiments with six donors d. Statistics: a, c Two-way ANOVA, followed by a Šidák’s test for multiple comparisons. b One-way ANOVA, followed by a Tukey’s test for multiple comparisons. d Two-tailed paired t test. The data are presented as mean ± SD. Only p values <0.05 are shown.

Finally, we investigated whether tissue glucose levels are dynamically regulated in acute allergic skin inflammation, in which T_H9 cells have been shown to highly express IL-9^{9, 31}. To this end, we determined glucose levels in the interstitial fluid of tissue homogenates from non-lesional and lesional ACD skin 48 h post allergen application, respectively. The interstitial fluid of the lesional skin contained higher glucose levels than the matched non-lesional skin samples (Fig. 6d), which suggests that IL-9-related human allergic skin inflammation is associated with changes in glucose availability in vivo.

Collectively, these observations suggest that paracrine IL-9 facilitates the proliferation of T cells by stimulating aerobic glycolysis through the induction of the lactate transporter MCT1, possibly contributing to the proliferation of IL-9R⁺ T_H cells in the high-glucose environment of ACD and acute allergic tissue inflammation. In addition, we found that PPAR-γ is a positive regulator of aerobic glycolysis in activated human T_H9 cells, which in turn, regulates the expression of IL-9 via mTORC1. Together, this suggests that PPAR-γ and IL-9 facilitate immunometabolic sensing of the tissue microenvironment (Fig. 6e).