Inducible costimulator expression regulates the magnitude of Th2-mediated airway inflammation by regulating the number of Th2 cells

Abstract

Background: Inducible Costimulator (ICOS) is an important regulator of Th2 lymphocyte function and a potential immunotherapeutic target for allergy and asthma. A SNP in the ICOS 5' promoter in humans is associated with increased atopy and serum IgE in a founder population and increased ICOS surface expression and Th2 cytokine production from peripheral blood mononuclear cells. However, it is unknown if increased ICOS expression contributes to disease progression or is a result of disease pathology.

Methodology/principal findings: We developed a mouse model in which ICOS surface expression levels are genetically predetermined to test our hypothesis that genetic regulation of ICOS expression controls the severity of Th2 responses in vivo. Using ICOS+/+ and ICOS+/- mice in a Th2 model of airway inflammation, we found that T cells from the ICOS+/- mice had reduced ICOS expression and decreased Th2-mediated inflammation in vivo. Although the activation status of the T cells did not differ, T cells isolated from the lungs and draining lymph nodes of ICOS+/- mice at the peak of inflammation produced less Th2 cytokines upon stimulation ex vivo. Using 4get mice, which express GFP upon IL-4 transcription, we determined that the decreased Th2 cytokines in ICOS+/- is due to reduced percentage of Th2 cells and not a defect in their ability to produce IL-4.

Conclusion: These data suggest that in both mice and humans, the level of ICOS surface expression regulates the magnitude of the in vivo Th2 response, perhaps by influencing Th2 differentiation.

Figure 1. ICOS^+/− T cells have decreased ICOS expression but equal activation status.

ICOS^+/+ (black lines) and ICOS^+/− (grey line) T cells were isolated and stimulated with anti-CD3 and anti-CD28 antibodies in media alone or in the presence of IL-4 or anti-IL-4 antibodies. After 48 hours the cells were removed and expression of the indicated surface markers were analyzed via flow cytometry. (A) ICOS expression on CD4⁺ T cells stimulated under the indicated conditions. (B) Expression of the indicated cell-surface markers after stimulation for 48 hours in media alone cultures.

Figure 2. ICOS cell-surface expression regulates the magnitude of a Th2 response in vivo.

ICOS^+/+ (black bars), ICOS^+/− (grey bars) and ICOS^−/− (white bars) mice were activated as indicated in Materials and Methods. (A) Total cells in BAL fluid were counted and percent CCR3⁺ eosinophils, CD4⁺ T cells and CD8⁺ T cells were analyzed via flow cytometry and used to calculate total cell numbers. (B) ICOS expression on BAL CD4⁺ T cells from ICOS^+/+ (black line), ICOS^+/− (grey line), ICOS^−/− (dotted line) is shown. (C) Serum IgE levels were analyzed via ELISA.

Figure 3. Defective Th2 cytokine production in ICOS^+/− mice.

(A) Four days after challenge the lungs were harvested and isolated cells were stimulated with anti-CD3 for 24 hours. Number of cells producing IL-4 and IL-5 were quantified via ELISPOT. (B) Mediastinal lymph nodes were stimulated with anti-CD3 for 24 hours, and the number of cells producing IL-4 and IL-5 were quantified via ELISPOT or (C) The cells were stimulated with SEA for 48 hours and cytokine supernatant levels were analyzed via ELISA.

Figure 4. B7RP-1 downregulation on APCs is regulated by ICOS expression.

(A) Cells were isolated from the mediastinal lymph node and spleen of ICOS^+/+ (Black line), ICOS^+/− (Grey line), and ICOS^−/− (dashed line) mice at the peak of inflammation. The B7RP-1 expression on CD11c^hi cells is shown. (B) Cells were isolated from the spleens of naïve mice (ICOS^+/+ n = 3; ICOS^−/− n = 4). The B7RP-1 MFI on the indicated cell types is shown. (C) Left- Cells were isolated from the spleens of naïve C57BL/6 (n = 3) and Rag^−/− (n = 3). The B7RP-1 MFI on CD11c^hi cells is shown. Right. T cells were isolated from naïve ICOS^+/+ and ICOS^−/− mice and transferred to Rag^−/− mice. Eight wks later the mice were sacrificed and B7RP-1 expression was analyzed via flow cytometry.

Figure 5. Decreased Th2 response in B7RP-1^+/− mice.

Splenocytes from Wild-type and B7RP-1^+/− mice were removed and single-cell suspensions were made. The cells were stained with B7RP-1 and either CD19 or CD11c. Black = WT Grey = B7RP-1^+/−. (B) B6. B7RP-1^+/+ (black bars), B6. B7RP-1^+/− (grey bars), and B6. B7RP-1^−/− (white bars) mice were sensitized and challenged as indicated in Materials and Methods. Total cells in BAL fluid were counted and percent CCR3⁺ eosinophils, CD4⁺ T cells and CD8⁺ T cells were analyzed via flow cytometry and used to calculate total cell numbers and normalized to the average of the wild-type value. ICOS expression on BAL CD4⁺ T cells isolated from the mediastinal lymph nodes of mice is shown. Serum IgE levels were analyzed via ELISA.

Figure 6. Reduced number of Th2 cells in the airways of ICOS^+/− mice.

(A) ICOS^+/+.4get (black bars), ICOS^+/−.4get (grey bars) mice were activated as indicated in Materials and Methods. Total cells in BAL fluid were counted and percent CCR3⁺ eosinophils, total CD4⁺ T cells, CD4⁺GFP⁺ and CD4⁺GFP⁻ T cells were analyzed via flow cytometry and used to calculate total cell numbers. (B) The percent of CD4⁺GFP⁺ cells from the indicated organs are shown. The results are from separate experiments that have been combined (ICOS^+/+ n = 7, ICOS^+/− n = 9). (C) CD4⁺GFP⁻ and CD4⁺GFP⁺ cells were sorted from the mediastinal lymph nodes and lungs of ICOS^+/+.4get and ICOS^+/−.4get mice. The cells were equalized and stimulated with anti-CD3 and anti-CD28 antibodies for 48 hours and the IL-4 production was analyzed via ELISPOT.