Inducible costimulator promotes helper T-cell differentiation through phosphoinositide 3-kinase

Abstract

The T-cell costimulatory receptors, CD28 and the inducible costimulator (ICOS), are required for the generation of follicular B helper T cells (T(FH)) and germinal center (GC) reaction. A common signal transducer used by CD28 and ICOS is the phosphoinositide 3-kinase (PI3K). Although it is known that CD28-mediated PI3K activation is dispensable for GC reaction, the role of ICOS-driven PI3K signaling has not been defined. We show here that knock-in mice that selectively lost the ability to activate PI3K through ICOS had severe defects in T(FH) generation, GC reaction, antibody class switch, and antibody affinity maturation. In preactivated CD4(+) T cells, ICOS delivered a potent PI3K signal that was critical for the induction of the key T(FH) cytokines, IL-21 and IL-4. Under the same settings, CD28 was unable to activate PI3K but supported a robust secondary expansion of T cells. Thus, our results demonstrate a nonredundant function of ICOS-PI3K pathway in the generation of T(FH) and suggest that CD28 and ICOS play differential roles during a multistep process of T(FH) differentiation.

Fig. 1.

Defective AKT and ERK activation by ICOS-YF. CD4⁺ T blasts were stimulated with antibodies against CD3 and/or ICOS, and the activation of AKT or MAPKs was measured by immunoblotting using phospho-specific antibodies. A representive of three independent experiments is shown.

Fig. 2.

ICOS-YF and KO mice have reduced levels of class-switched immunoglobulins in serum. Sera were obtained from nonimmunized mice of ICOS WT, YF, and KO mice at 8 weeks of age. Concentrations of IgM, G1, G2a, G2c, G3, and A were determined by isotype specific ELISA. Each data point represents a serum Ig level of an individual mouse (n = 10 WT, 14 YF, and 10 KO). *, P < 0.01.

Fig. 3.

ICOS-YF as well as KO mice have severely impaired humoral immune responses in the PP. GC B cells and T_FH cells in the PP were analyzed by flow cytometry. The (Top) two were gated on B220⁺ B cells and the (Bottom) two on CD4⁺ T cells. Numbers represent mean ± SD of data pooled from four mice per genotype.

Fig. 4.

Impaired Ab responses in ICOS-YF and ICOS-KO mice upon immunization. (A) Defective GC reaction. Cryosections of spleens from mice immunized with NP₁₆-CGG/alum 12 days before were stained with PNA. (B) Decreased GC B cells and T_FH cells in ICOS-YF and KO mice. Mice were immunized with NP₁₆-CGG/alum, and the splenocytes were analyzed 12 days later. Percentages represent mean ± SD of data from three mice per genotype. A representative of two independent experiments. (C) Impaired class switch. Sera were prepared from immunized mice at day 11 (1°) or day 7 (2°) postinjection, and antigen-specific IgG1 were measured by ELISA using NP₃₃-BSA vs. NP₃-BSA. Numbers represent mean ± SD of data from six mice per genotype. A representative of two independent experiments. (D) Defective Ab affinity maturation. Mice were immunized at day 0 and boost injected at day 30. Serum samples were prepared at day 11 after primary injection or day 7 after secondary injection, and the anti-NP IgG1 was measured after differential washes with increasing concentrations of NaSCN solution. Each histogram represents mean ± SD of data from six mice per genotype. A representative of two independent experiments.

Fig. 5.

ICOS-YF and ICOS-KO CD4⁺ T cells have defects in IL-21 and IL-4 expression. (A) ICOS induces IL-21 and IL-4 in a PI3K-dependent manner. Preactivated CD4⁺ T cells were restimulated with anti-CD3 plus hamster IgG (H), anti-ICOS (I), or anti-CD28 (28) followed by goat anti-hamster IgG for 6 h without or with LY 294002. The cytokine mRNA levels in the restimulated cells were analyzed by qPCR. A representative of three independent experiments. (B) Abrogation of IL-21 and IL-4 induction in ICOS-YF T cells. Cytokine gene expression was analyzed in WT, YF, and KO T cells as described in A. Each data point represents fold increase over hamster IgG control. Data pooled from six independent experiments (n = 5–6 WT, 2–3 YF, and 2–3 KO). *, P < 0.02.