Thymic stromal lymphopoietin drives the development of IL-13+ Th2 cells

Abstract

T helper 2 (Th2) cells are pivotal in the development of allergy. Allergen exposure primes IL-4⁺ Th2 cells in lymph node, but production of effector cytokines including IL-5 and IL-13 is thought to require additional signals from antigen and the environment. Here we report that a substantial proportion of naive CD4⁺ T cells in spleen and lymph node express receptors for the epithelium-derived inflammatory cytokine thymic stromal lymphopoietin (TSLP). Culture of naive CD4⁺ T cells in anti-(a)CD3, aCD28, and TSLP-supplemented Th2 conditions enabled the development of a unique population of IL-13-single positive (IL-13-SP) CD4⁺ T cells; TSLP and Th2 conditions were both required for their development. Sorting experiments revealed that IL-13-SP Th2 cells originated from IL-4-negative precursors and coexpressed transcripts for the Th2 cytokines IL-5 and IL-9. In vivo, high TSLP levels acted directly on CD4⁺ T cells to induce the development of IL-13-SP and IL-4⁺IL-13⁺ double-positive populations in lymph node. These cells were phenotypically similar to Th2 effector cells and were CXCR5^lowPD1^low and expressed low levels of Bcl6 and Il21 transcripts and high levels of Gata3, Il3, and Il5 Our findings suggest a role of TSLP in directly promoting Th2 cell effector function and support the notion of TSLP as a key driver of Th2 inflammation.

Keywords: IL-13; IL-4; TSLP; Th2 cells; effector T cells.

Fig. 1.

Naive and antigen-experienced CD4⁺ T cells express both subunits of the TSLP receptor. CD4⁺ T cells from spleen and pooled skin-dLN of C57BL/6 or TSLPR-KO mice were examined for coexpression of TSLPR and IL7R by flow cytometry. (A) Gating strategy for splenic CD4⁺ T cells. (B) Expression of TSLPR and IL7R in splenic CD4⁺ T cell subsets. (C) Expression of TSLPR and IL7R in skin-dLN CD4⁺ T cell subsets. Bar graphs show mean and SD for five mice/group; each dot corresponds to one mouse. Data refer to one of three repeat experiments that gave similar results. FMO, fluorescence minus one control.

Fig. 2.

TSLP elicits IL-13DR expression by naive CD4⁺ T cells cultured in Th2 conditions. Splenic naive CD4⁺ T cells from 4C13R reporter mice were sorted and cultured in Th2 or Th0 conditions in the presence or absence of TSLP. Cells were harvested after 1–5 d in culture and analyzed for expression of IL-4-AC only (IL-4AC SP), IL-13-DR only (IL-13DR SP), or both. Data refer to one of at least three repeat experiments that gave similar results. ***P < 0.001; **P < 0.01; *P < 0.05.

Fig. 3.

IL-13DR-expressing Th2 cells arise from an IL-4AC-negative population in the presence of TSLP. Naive CD4⁺ T cells were purified and cultured as in Fig. 2. On day 2, cells were harvested, sorted into IL-4AC⁺ or IL-4AC⁻, and cultured in the same media for up to 3 d before analysis of reporter expression. (A) Expression of IL-4AC in representative presort and sorted populations on day 2. (B) Expression of IL-4AC and IL-13DR in recultured populations on day 5. (C) Time-course analysis of IL-4AC and/or IL-13DR reporter expression in culture. Graphs show mean ± SD for three independent cultures/group. Data refer to one of three repeat experiments that gave similar results. **P < 0.01.

Fig. 4.

Culture in Th2 conditions and TSLP generates a population of Th2 cells that express IL-13, IL-5, and IL-9. Naive CD4⁺ T cells were purified and cultured as in Fig. 2. (A) Cultured cells were harvested on day 5 and sorted into DN, IL-4AC SP (AC⁺), or where applicable, IL-13DR SP (DR⁺) for RT-qPCR analysis. Cytokine expression data are normalized to Gapdh and relative to Th2 DN cells (left column). (B and C) On day 3–5, T cells were harvested, restimulated with aCD3/aCD28, and stained for intracellular IL-13 and IL-9 (B), or IL-13 and IL-5 (C). Graphs show mean ± SD for three independent cultures/group. Data refer to one of two repeat experiments that gave similar results. **P < 0.01; *P < 0.05.

Fig. 5.

High TSLP levels promote the development of CD4⁺ IL-13DR⁺ cells with a PD1^lowCXCR5^low phenotype in vivo. C57BL/6 and 4C13R mice were treated with HDM or MC903. Tissues were collected for analysis at the indicated points. (A) Expression of Tslp transcripts in the epidermal layers of C57BL/6 mice. Expression is normalized to 18S RNA and relative to day 0. (B) Serum TSLP in MC903-treated C57BL/6 mice. Data are from one of two repeat experiments that gave similar results; each dot corresponds to one mouse. (C and D) Phenotype of reporter-expressing CD4⁺CD44^high T cells in the dLN of HDM-treated (C) or MC903-treated (D) 4C13R mice on day 7. No reporter expression was detected in the CD44-low population. (E) Total dLN cellularity and percentage of reporter-expressing CD4⁺CD44^high T cells in 4C13R mice treated with HDM or MC903. (F) Numbers of reporter-expressing CD4⁺CD44^high T cells with a “Tfh” or “Th2eff” phenotype in the dLN of 4C13R mice treated with HDM or MC903. Phenotype was determined as in C and D. Line graphs show mean ± SD for six mice/group from one of two repeat experiments that gave similar results. P values in F refer to the comparisons of HDM to MC903. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05.

Fig. 6.

The development of CD4⁺ IL-13DR cells in vivo requires expression of TSLPR. (A) 4C13R reporter mice bred onto a C57BL/6 or TSLPR-KO background were treated with HDM or MC903. The dLN were harvested on day 7, and reporter expression was evaluated by flow cytometry as in Fig. 5 C and D. (B) CD4-enriched cells from CD45.1⁺2⁺ WT mice and CD45.2⁺ TSLPR-KO mice, both expressing the 4C13R double reporter, were transferred into WT CD45.1⁺ recipient mice at a ∼1:1 ratio. Recipient mice were treated with EtOH or MC903, and dLN were harvested on day 7 for analysis of reporter expression. Dot plots show concatenated data from five mice. (C) Ratios of adoptively transferred TSLPR-KO to WT cells in the indicated populations; P values refer to the comparison with the MC903-tot group. Bar graphs show mean and SD from one of two to three repeat experiments that gave similar results; each dot represents one mouse. ***P < 0.001; **P < 0.01; *P < 0.05; ns: not significant.

Fig. 7.

CD4⁺ IL-13DR SP LN T cells express Th2 cytokine transcripts in vivo. 4C13R mice were treated with HDM or MC903; untreated mice (NT) were used as controls. dLN were harvested on day 7, and CD4⁺CD44^high T cells were sorted according to reporter expression as indicated, without in vitro restimulation. Expression of the indicated transcripts is shown relative to Gapdh and the IL-13DR SP population. Data are from one of three repeat experiments that gave similar results; each dot refers to one mouse. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns: not significant.