Distinct regulation of interleukin-17 in human T helper lymphocytes

Abstract

Objective: Interleukin-17 (IL-17)-producing T helper cells have been proposed to represent a separate lineage of CD4+ cells, designated Th17 cells, which are regulated by the transcription factor retinoic acid-related orphan receptor gammat (RORgammat). However, despite advances in understanding murine Th17 differentiation, a systematic assessment of factors that promote the differentiation of naive human T cells to Th17 cells has not been reported. The present study was undertaken to assess the effects on naive human CD4+ T cells of cytokines known to promote murine Th17 cells.

Methods: Human naive and memory CD4+ T cells isolated from peripheral blood were activated and cultured with various cytokines. Cytokine production was measured by enzyme-linked immunosorbent assay and flow cytometry. Messenger RNA was measured by quantitative polymerase chain reaction.

Results: In response to anti-CD3/anti-CD28 stimulation alone, human memory T cells rapidly produced IL-17, whereas naive T cells expressed low levels. Transforming growth factor beta1 and IL-6 up-regulated RORgammat expression but did not induce Th17 differentiation of naive CD4+ T cells. However, IL-23 up-regulated its own receptor and was an important inducer of IL-17 and IL-22.

Conclusion: The present data demonstrate the differential regulation of IL-17 and RORgammat expression in human CD4+ T cells compared with murine cells. Optimal conditions for the development of IL-17-producing T cells from murine naive precursors are ineffective in human T cells. Conversely, IL-23 promoted the generation of human Th17 cells but was also a very potent inducer of other proinflammatory cytokines. These findings may have important implications in the pathogenesis of human autoimmunity as compared with mouse models.

Figure 1. Human memory helper T cells produce significantly higher levels of IL-17

(a,b). Isolated human naïve and memory CD4⁺ T cells were stimulated with plate-bound anti-CD3 and anti-CD28 for 24 hours. IL-17 production was detected by ELISA immunoassay kits. A representative result is shown in panel a and data from five healthy donors are shown in panel b, where the horizontal line indicates geometric mean. Statistical significance was calculated based on geometric mean using paired t-test.

Figure 2. Optimal conditions for murine Th17 differentiation do not enhance Th17 differentiation of human naïve CD4⁺ T cells

a. Naïve CD4⁺ T cells from normal donors (n = 5) were activated with plate-bound anti-CD3 and anti-CD28 and cultured for two days with IL-2 (Th0) or optimal polarizing conditions for murine Th17 differentiation (TGF-β1+IL-6+anti-IFN-γ+anti-IL-4). IL-17 secretion in cell culture supernatants was detected by ELISA. Statistical significance was calculated using the paired Student’s t-test. b. Naïve CD4⁺ T cells were activated and cultured under Th0 or Th17 conditions for 7 days. At day 7, cells were activated with anti-CD3 and -CD28 and cultured under Th0 or Th17 conditions for another 7 days. IL-17 production in cell culture supernatants was measured by ELISA at various timepoints. c. Quantitation of IL-17- and FOXP3-producing cells after stimulation of naïve CD4⁺ T cells under Th0 or “Th17” conditions, as determined by intracellular cytokine staining and flow cytometry. d. Quantitation of IL-17- and IFN-γ-producing cells at 24 h and 7 days post-stimulation was determined by intracellular cytokine staining and flow cytometry.

Figure 3. Plasticity of human IL-17 producing T cells

a. Isolated naïve CD4₊ T cells were cultured under Th0, Th1, Th2 and Th17 conditions for 7 days, and IL-17 protein production was measured by ELISA directly or after restimulated with anti-CD3 and anti-CD28 for an additional 24 h. b. Isolated memory CD4⁺ T cells from two different donors were activated by plate-bound anti-CD3 and anti-CD28 for indicated time. IFN-γ and IL-17 producing cells were analyzed by intracellular cytokine staining using flow cytometry.

Figure 4. IL-23 upregulates its receptor and enhances IL-17 production in human CD4⁺ T cells

(a). IL-23R mRNA expression in isolated naïve or memory CD4⁺ T cells was analyzed by real-time quantitative PCR. (b). Naïve CD4⁺ T cells were activated with plate-bound anti-CD3 and anti-CD28 and cultured under Th0, Th1, Th2, Th17 polarizing conditions or with IL-23 (10 ng/ml), anti-IFN-γ (10 μg/ml) and anti-IL-4 (10 μg/ml) for 2 days. IL-23R mRNA expression was detected by real time quantitative PCR. (c). Naïve CD4⁺ T cells were cultured under Th0 or Th17 conditions in the presence or absence of IL-23 and anti-IFN-γ and anti-IL-4 or Th1 conditions for 14 days. At day 14, cells were restimulated with anti-CD3/CD28 for 24 hours. IL-17 in cell culture supernatants was detected by ELISA. The figure depicts the mean of data from three separate donors analyzed in duplicate. Statistical significance was determined using the paired Student’s t-test, n=6. (d). Naïve or memory CD4⁺ T cells were activated by plate-bound anti-CD3 and anti-CD28 in the presence or absence of IL-23 for 24h. IL-17 was measured by ELISA.

Figure 5. Regulation of RORγt in human CD4⁺ T cells

Naïve and memory CD4⁺ T cells were cultured under Th0 or Th17 conditions with or without IL-23 for 48 h. RORγt (a) and IL-17 (b) mRNA were detected with real-time quantitative PCR. (c). Naïve CD4⁺ T cells were cultured under Th0, Th1, or Th17 conditions or with IL-23 and anti-IFN-γ and anti-IL-4 for 14 days as indicated. At day 14, cells were restimulated with anti-CD3 and anti-CD28 for 24 h. RORγt mRNA expression was analyzed by quantitative PCR. The figure depicts data from three separate donors assayed in duplicate. Statistical significance was calculated based on paired Student’s t-test, n=6.

Figure 6. IL-23 enhances IL-22 production

(a). Human naïve or memory CD4⁺ T cells were stimulated with plate-bound anti-CD3 and anti-CD28 in the presence or absence of IL-23 for 24 hours. IL-22 protein in cell culture supernatants was detected by ELISA. The data represent the mean of four independent experiments. Statistical significance was calculated based on paired Student’s t-test. (b). Naïve CD4⁺ T cells were cultured under Th0 or Th17 conditions or with IL-23 for two days. IL-22 protein production in cell culture supernatants was measured by ELISA (n = 6) (c). Naïve CD4⁺ T cells were cultured under Th0, Th1, Th2 or Th17 polarizing conditions, or with IL-23 for 14 days and IL-22 mRNA was detected by Taqman at the indicated timepoints. (d). Naïve CD4⁺ T cells were activated with plate-bound anti-CD3 and anti-CD28 in the presence or absence of IL-23 and anti-IFN-γ and anti-IL-4 for two days before measuring IL-22 protein levels in culture supernatants by ELISA. The horizontal line indicates geometric mean of each stimulation. Statistical significance was calculated based on geometric means using the paired t-test.