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. 2010 Oct 27;2(55):55ra78.
doi: 10.1126/scitranslmed.3000448.

The inducible costimulator (ICOS) is critical for the development of human T(H)17 cells

Chrystal M Paulos  1 , Carmine Carpenito, Gabriela Plesa, Megan M Suhoski, Angel Varela-Rohena, Tatiana N Golovina, Richard G Carroll, James L Riley, Carl H June
Affiliations

The inducible costimulator (ICOS) is critical for the development of human T(H)17 cells

Chrystal M Paulos et al. Sci Transl Med. .

Abstract

Human T helper 17 (T(H)17) cells regulate host defense, autoimmunity, and tumor immunity. Although cytokines that control human T(H)17 cell development have been identified, the costimulatory molecules important for T(H)17 cell generation are unknown. Here, we found that the inducible costimulator (ICOS) was critical for the differentiation and expansion of human T(H)17 cells. Human cord blood contained a subset of CD161(+)CD4(+) T cells that were recent emigrants from the thymus, expressed ICOS constitutively, and were imprinted as T(H)17 cells through ICOS signaling. ICOS stimulation induced c-MAF, RORC2, and T-bet expression in these cells, leading to increased secretion of interleukin-21 (IL-21), IL-17, and interferon-γ (IFN-γ) compared with cells stimulated with CD28. Conversely, CD28 ligation abrogated ICOS costimulation, dampening RORC2 expression while promoting the expression of the aryl hydrocarbon receptor, which led to reduced secretion of IL-17 and enhanced production of IL-22 compared with cells stimulated with ICOS. Moreover, ICOS promoted the robust expansion of IL-17(+)IFN-γ(+) human T cells, and the antitumor activity of these cells after adoptive transfer into mice bearing large human tumors was superior to that of cells expanded with CD28. The therapeutic effectiveness of ICOS-expanded cells was associated with enhanced functionality and engraftment in vivo. These findings reveal a vital role for ICOS signaling in the generation and maintenance of human T(H)17 cells and suggest that components of this pathway could be therapeutically targeted to treat cancer or chronic infection and, conversely, that interruption of this pathway may have utility in multiple sclerosis and other autoimmune syndromes. These findings have provided the rationale for designing new clinical trials for tumor immunotherapy.

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Figures

Fig. 1.
Fig. 1.
Distinct expression and function of ICOS and CD28 on human CD4+ T cell subsets. (A) The expression of ICOS and CD28 costimulatory molecules was assessed on resting human peripheral blood CD4+ T cell subsets, consisting of CXCR3+CCR4−CCR6+ TH1, CCR4+CXCR3−CCR6− TH2, CCR4+CCR6+ TH17, CD25+CD127loFoxP3+ Treg, and CXCR5+CD45RO+ TFH cells. (B) Flow cytometric quantification of ICOS and CD28 on different subsets from several normal donors (n = 7). Horizontal bars indicate mean. ns, not significant. (C) Cytokines IL-2 (i), IL-4 (ii), IFN-γ (iii), IL-10 (iv), IL-22 (v), IL-17A (vi), IL-17F (vii), CCL20 (viii), and IL-21 (ix) secreted from various sorted cells activated with antibodies to CD3/CD28 or CD3/ICOS beads and measured on day 3 by ELISA. Representative of three experiments. Statistics were corrected for multiple comparisons with the ANOVA Scheffé test. TFH, follicular helper T.
Fig. 2.
Fig. 2.
ICOS augments cytokine production by human TH17 cells. (A) IL-17F production was assessed by peripheral blood CD4+ T cells differentiated to a TH17 phenotype with TH17-polarizing conditions (IL-6, IL-1β, IL-23, neutralizing IFN-γ, and neutralizing IL-4 antibodies in serum containing TGF-β, a cytokine required for inducing TH17 differentiation) and activated with either aAPCs expressing CD86, CD80, CD70, ICOSL, OX40L, or 4-1BBL or with beads bearing antibodies to CD3 and CD28 on day 3 by ELISA. (B) IL-17F production was assessed by peripheral blood CD4+ T cells cultured with or without TH17-polarizing conditions and activated with aAPC engineered to express ICOSL or with beads bearing antibodies to CD3/ICOS on day 3. (C to G) Using ELISA or reverse transcription PCR (RT-PCR), we measured (C) IL-17F, (D) IL-17A, (E) IL-2, (F) IL-22, and (G) IL-10 secretion or expression by TH17-polarized CD4+ T cells activated with beads bearing antibodies to CD3, CD28, and/or ICOS on day 3. Representative of two experiments.
Fig. 3.
Fig. 3.
ICOS is critical for the expansion of human TH17 cells. (A and B) The frequency (A) and absolute number (B) of CCR4+CCR6+CD4+ T cells over time were assessed by flow cytometry from peripheral blood CD4+ T cells cultured in TH17-polarizing conditions and activated with antibodies to CD3/CD28 or CD3/ICOS beads. (C) CD27 and CD62L expression was measured on day 10 on these cells with flow cytometry. (D) On the days indicated, CD28- or ICOS-engaged TH17-polarized CD4+ T cells were stimulated with PMA-ionomycin and the frequency of cells secreting IL-17A and IFN-γ was assessed via flow cytometry. (E) The frequency of CD28- or ICOS-engaged TH17-polarized cells coproducing IL-17A and/or IFN-γ was determined at the end of their primary expansion (ranging from days 9 to 14) in several different normal donors (n = 8). (F and G) RORC2 and T-bet expression in these treated cells was measured with RT-PCR on days 3 and 10. Representative of two to three experiments.
Fig. 4.
Fig. 4.
ICOS drives rapid TH17 cell differentiation from naive UCB CD4+ T cells. (A to C) UCB CD45RA+CD25−CD4+ T cells were cultured with TH17-polarizing conditions and expanded with antibodies to CD3/CD28, CD3/ICOS, or CD3/CD28/ICOS beads. Starting on day 3, IL-2 (50 lU/ml) was added to the cultures. Cultures were stimulated with PMA-ionomycin (IONO) and the intracellular expression of IL-17A, IFN-γ, IL-2, and TNF-α and the extracellular expression of IL-23R and CD161 were assessed on day 11. Cells from (A) to (C) were reactivated with antibodies to CD3-coupled beads bearing antibodies to CD28 and/or ICOS. (D to F) Cultures were restimulated with PMA-ionomycin and the intracellular expression of IL-17A, IFN-γ, IL-2, and TNF-α and the extracellular expression of IL-23R and CD161 were assessed on day 18. Representative of two experiments.
Fig. 5.
Fig. 5.
CD28 and ICOS differentially regulate c-MAF, RORC2, and T-bet expression in UCB TH17 cells. UCB CD4+T cells were cultured in TH17-polarizing conditions and expanded with antibodies to CD3/CD28 or CD3/ICOS beads. IL-2 (50 IU/ml) was added on day 3. (A and B) On day 5, mRNA expression of c-MAF and IL-21 in CD28- or ICOS-stimulated cells was measured by RT-PCR. (C) On day 5, IL-17F production in CD28-stimulated cells cultured with exogenous IL-21 and IL-2 neutralization was measured by ELISA. (D to L) On the days indicated, RORC2, T-bet, FoxP3, AHR, IL-22, IL-10, and IL-17A production in CD28- or ICOS-stimulated cells was measured by flow cytometry and RT-PCR. Representative of two to three experiments.
Fig. 6.
Fig. 6.
Human TH17 cells originate from ICOS+CD161+CD4+T cell precursors. (A) CD45RA, CD31, CD127, CD62L, and CD27 expression was assessed on ICOS+CD161+CD4+ and ICOS−CD161+CD4+ T cells from the UCB via flow cytometry. (B) IL-17F, CCL20, IFN-γ, IL-4, IL-22, and IL-10 secretion by sorted ICOS+CD161+CD4+ and ICOS−CD161+CD4+ T cells cultured with TH17-polarizing conditions and expanded with antibodies to CD3/CD28 or CD3/ICOS beads was assessed on day 4 by ELISA. (C) The frequency and absolute number of CD161+ cells cultured with TH17-polarizing conditions and expanded with antibodies to CD3/CD28- or CD3/ICOS-coated beads was determined on day 4 or on the days indicated, respectively. (D) RORC2, IL-23R, AHR, and FoxP3 mRNA expression in sorted ICOS+CD161+CD4+ and ICOS−CD161+CD4+ T cells cultured with TH17-polarizing conditions and expanded with antibodies to CD3/CD28- or CD3/ICOS-coated beads was assessed on day 7 by RT-PCR. (E) On day 7, ICOS+CD161+CD4+ and ICOS−CD161+CD4+ T cells cultured in media alone or in TH1-, TH2-, TH17-, and Treg-polarizing conditions and expanded with antibodies to CD3/CD28- or CD3/ICOS-coated beads were then stimulated with PMA-ionomycin, and IL-17A secretion was assessed by flow cytometry. Representative of two to three experiments.
Fig. 7.
Fig. 7.
ICOS augments T cell-mediated tumor immunity. As shown schematically, human CD4+ and CD8+ T cells were stimulated with antibodies to CD3/CD28 or CD3/ICOS beads and cultured with or without TH17-polarizing conditions. One day later, bead-activated T cells were genetically redirected with a CAR that binds mesothelin. After their primary expansion, the genetically redirected cells (two administrations, 8 × 106 cells total) were infused into mice bearing a large human mesothelin (M108) tumor preestablished for 61 days (n = 8 mice per group). (A to D) Tumor growth was measured in mice infused with genetically redirected cells expanded with the ICOS or CD28 signal with or without TH17-polarizing conditions. Tumor growth was analyzed with a linear mixed-effects model and by applying a conservative Bonferroni correction approach (mean ± SEM). (E) Redirected T cells were isolated from the mouse spleens (on day 43) and cultured with irradiated aAPCs bearing mesothelin. IL-17A and IFN-γ secretion was analyzed by flow cytometry 24 hours later. (F) The absolute number of CD4+ and CD8+ T cells was determined in the blood and spleen on days 21 and 43, respectively. Representative of two experiments.
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References

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