Phosphatidylinositol 3-kinase-independent signaling pathways contribute to ICOS-mediated T cell costimulation in acute graft-versus-host disease in mice - PubMed (original) (raw)

Phosphatidylinositol 3-kinase-independent signaling pathways contribute to ICOS-mediated T cell costimulation in acute graft-versus-host disease in mice

Jun Li et al. J Immunol. 2013.

Abstract

We and others have previously shown that ICOS plays an important role in inducing acute graft-versus-host disease (GVHD) in murine models of allogeneic bone marrow transplantation. ICOS potentiates TCR-mediated PI3K activation and intracellular calcium mobilization. However, ICOS signal transduction pathways involved in GVHD remain unknown. In this study, we examined the contribution of ICOS-PI3K signaling in the pathogenic potential of T cells using a knock-in mouse strain, ICOS-YF, which selectively lost the ability to activate PI3K. We found that when total T cells were used as alloreactive T cells, ICOS-YF T cells caused less severe GVHD compared with ICOS wild-type T cells, but they induced much more aggressive disease than ICOS knockout T cells. This intermediate level of pathogenic capacity of ICOS-YF T cells was correlated with similar levels of IFN-γ-producing CD8 T cells that developed in the recipients of ICOS-WT or ICOS-YF T cells. We further evaluated the role of ICOS-PI3K signaling in CD4 versus CD8 T cell compartment using GVHD models that are exclusively driven by CD4 or CD8 T cells. Remarkably, ICOS-YF CD8 T cells caused disease similar to ICOS wild-type CD8 T cells, whereas ICOS-YF CD4 T cells behaved very similarly to their ICOS knockout counterparts. Consistent with their in vivo pathogenic potential, CD8 T cells responded to ICOS ligation in vitro by PI3K-independent calcium flux, T cell activation, and proliferation. Thus, in acute GVHD in mice, CD4 T cells heavily rely on ICOS-PI3K signaling pathways; in contrast, CD8 T cells can use PI3K-independent ICOS signaling pathways, possibly through calcium.

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Conflict of interest statement

Disclosures

The authors have no financial conflicts of interest.

Figures

FIGURE 1

ICOS-YF T cells retain the ability to induce acute GVHD. Lethally irradiated (800 cGy) BALB/c mice were transplanted with 5 × 106 B6 WT TCD-BM without or with 1 × 106 purified total T cells from ICOS-WT, YF, or KO mice in B6 background. Overall survival (A) and the percentage of original body weight over time (B) are depicted. The data are accumulated from three replicate experiments with 15–18 mice per group. BMT was set up as in (A) and (B), and liver, lung, small intestine, and colon samples were collected at 14 d after BMT for pathologic analysis (C). Pathological scores are presented individually using a semiquantitative scoring system, as described in Materials and Methods. The data are accumulated from two independent experiments with eight mice per group.

FIGURE 2

ICOS-mediated signals affect T cell migration and infiltration. Lethally irradiated (800 cGy) BALB/c mice were transplanted with 5 × 106 WT TCD-BM from Ly5.1+ B6 donors without or with 1 × 106 purified total T cells from ICOS-WT, YF, or KO mice in B6 background (Ly5.1−). Recipient spleen, liver, and small intestine were collected at 14 d after BMT. Mononuclear cells isolated from these organs were counted and stained for expression of H-2Kb (donor marker), Ly5.1 (marker for donor BM-derived cells), CD4, CD8, and α4β7. Absolute numbers of donor CD4 (CD4+H-2KbLy5.1−) and CD8 (CD8+H-2KbLy5.1−) cells were calculated and displayed in spleen (A and B), liver (D and E), and small intestine (G and H). Percentages of α4β7+ cells gated on donor CD4 or CD8 cells are shown in spleen (C) and liver (F). The score of lymphocyte infiltration was shown in recipient small intestine (I). The data are obtained from one of two replicate experiments with four mice per group in each experiment (A–H), but from two pooled experiments with eight mice per group (I).

FIGURE 3

Cytokine profiles induced by ICOS mutant T cells. BMT was set up as in Fig. 1. Peripheral blood and spleens were collected from each recipient at 14 d after BMT. Spleen cells were stained for expression of H-2Kb (donor marker), CD4, CD8, and intracellular IFN-γ. (A) Percentages of IFN-γ+ cells are shown on gated donor CD4 or CD8 cells (B). The levels of IFN-γ (C) and TNF-α (D) in the serum of the recipient mice were measured using cytometric bead array kits. The data are accumulated from two independent experiments with four to eight mice per group.

FIGURE 4

ICOS-YF CD4 T cells are severely impaired in GVHD pathogenic potential. Lethally irradiated (800 cGy) BALB/c mice were transplanted with 5 × 106 WT B6 TCD-BM without or with 1 × 106 purified CD4 T cells from ICOS-WT, YF, or KO mice in B6 background. Overall survival (A) and the percentage of original body weight over time (B) are depicted. The data are accumulated from two replicate experiments with 10–12 mice per group.

FIGURE 5

ICOS-YF CD8 T cells induce moderate GVHD. Lethally irradiated (800 cGy) BALB/c mice were transplanted with 5 × 106 TCD-BM alone or after supplementing with 0.2 × 106 WT CD4 T cells plus 1 × 106 CD8 T cells from ICOS-WT, YF, or KO mice. Overall survival (A) and the percentage of original body weight over time (B) are depicted. The data are accumulated from two independent experiments with 9–10 mice per group. For bm1 model, sublethally irradiated (600 cGy) B6.bm1 mice were transplanted with 1 × 106 CD8 T cells from ICOS-WT, YF, or KO mice. Overall survival (C) and the percentage of original body weight over time (D) are depicted. The data are accumulated from three independent experiments with 14–16 mice per group.

FIGURE 6

ICOS-YF can potentiate TCR-mediated calcium flux in CD8 T cells. Preactivated CD8 T cells were prepared from ICOS-WT, YF, and KO mice, and their ICOS-PI3K signaling (A) and ICOS-calcium signaling (B) capacities were examined, as described in Materials and Methods. Data shown are representative of three independent experiments.

FIGURE 7

Contribution of PI3K signaling to ICOS costimulation in T cell activation and proliferation in vitro. Purified T cells from ICOS-WT, YF, or KO mice were labeled with CFSE and stimulated with immobilized anti-CD3 at 0.5 μg/ml in the presence of anti-ICOS at the concentrations indicated. Two days after stimulation, cells were harvested and stained for surface expression of CD4, CD8, and CD25, and intracellular expression of IFN-γ. (A) For WT T cells, CD4 and CD8 expression on gated live cells (left panels); CFSE profile and CD25 expression on gated live CD4 (middle) or CD8 cells (right). (B) The experiment was done as in (A); CFSE profile and CD25 expression are presented. Percentages of CD25+ and CFSE-diluted cells (upper left quadrant) are depicted on gated live CD4 (C) or CD8 (D) cells of ICOS-WT, YF, or KO genotype. Percentages of IFN-γ+ and CFSE-diluted cells are depicted on gated live CD4 (E) or CD8 (F) cells of ICOS-WT, YF, or KO genotype. Data shown are from a representative experiment of three independent experiments.

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Phosphatidylinositol 3-kinase-independent signaling pathways contribute to ICOS-mediated T cell costimulation in acute graft-versus-host disease in mice - PubMed (original) (raw)