Involvement of HAb18G/CD147 in T cell activation and immunological synapse formation

Abstract

HAb18G/CD147, a glycoprotein of the immunoglobulin super-family (IgSF), is a T cell activation-associated molecule. In this report, we demonstrated that HAb18G/CD147 expression on both activated CD4(+) and CD8(+) T cells was up-regulated. In vitro cross-linking of T cells with an anti-HAb18G/CD147 monoclonal antibody (mAb) 5A12 inhibited T cells proliferation upon T cell receptor stimulation. Such co-stimulation inhibited T cell proliferation by down-regulating the expression of CD25 and interleukin-2 (IL-2), decreased production of IL-4 but not interferon-γ. Laser confocal imaging analysis indicated that HAb18G/CD147 was recruited to the immunological synapse (IS) during T cell activation; triggering HAb18G/CD147 on activated T cells by anti-HAb18G/CD147 mAb 5A12 strongly dispersed the formation of the IS. Further functional studies showed that the ligation of HAb18G/CD147 with mAb 5A12 decreased the tyrosine phosphorylation and intracellular calcium mobilization levels of T cells. Through docking antibody-antigen interactions, we demonstrated that the function of mAb 5A12 is tightly dependent on its specificity of binding to N-terminal domain I, which plays pivotal role in the oligomerization of HAb18G/CD147. Taken together, we provide evidence that HAb18G/CD147 could act as a co-stimulatory receptor to negatively regulate T cell activation and is functionally linked to the formation of the IS.

Fig 1

Measurement of HAb18G/CD147 expression on resting and activated CD²⁴⁺ and CD⁸⁺ T cell subsets. Purified resting T cells and activated T cells were stained with PE- conjugated anti-CD147, FITC-conjugated anti-CD4 or CD8, and were analysed by flow cytometry. (A) Histogram shows expression of CD147 on resting and activated CD²⁴⁺ T cells. (B) Histogram shows expression of CD147 on resting and activated CD⁸⁺ T cells. These data are representative of a minimum of three separate experiments.

Fig 2

HAb18G/CD147 accumulates to the cap and lipid rafts in T cells upon TCR stimulation confirmed by confocal microscopy and Western blot. (A, B, C, D) HAb18G/CD147 co-caps with CD4/CD8/CD48/GM1. T cells were prepared and stained as described previously in ‘Materials and methods’. HAb18G/CD147, TCR co-receptor CD4 and CD8, lipid raft marker CD48 and GM1 diffusely distribute on the cell membrane of resting T cells (upper panel of Fig. 2A–D), whereas stimulation results in translocation of these molecules to the cap induced by TCR/CD3 (lower panel of Fig. 2A–D), scale bar, 5 μm. (E) HAb18G/CD147 is recruited to lipid rafts upon T cell activation. Lipid rafts were isolated as described previously in ‘Materials and methods’. Sucrose fractions were analysed by Western blot with HAb18, anti-Fyn and anti-Rab5 antibodies. Detergent-insoluble membrane fractions containing isolated rafts were identified by the presence of the raft marker protein Fyn. Non-raft components of the membrane (e.g. Rab5) were purified in the heavy fraction (HF). HAb18G/CD147 appeared constitutively in the HF (7–9) with the non-raft marker Rab5 in resting T cells, a significant fraction of HAb18G/CD147 was localized within raft fractions (1–4) after T cell activation.

Fig 3

HAb18G/CD147 together with CD48 and GM1 are enriched into the IS. Raji cells were labelled with the blue fluorescent cytoplasmic probe CMAC first. Jurkat cells were then incubated with CMAC-labelled and 1 μg/ml of SEB-loaded (or not) Raji cells. Conjugates were then plated onto cover slip, fixed, stained with antibodies to HAb18G/CD147 and to CD48 (Fig. 3A) or GM1 (Fig. 3B), and visualized by confocal fluorescence microscopy. The red and green images represent the localization and distribution of HAb18G/CD147 and CD48/GM1, respectively. Raji cells are shown in blue. Scale bar, 5 μm.

Fig 4

Influence of HAb18G/CD147 mAbs on T cell proliferation. Proliferation was determined on day 4 following stimulation as described in ‘Materials and methods’. This figure shows the [³H]-thymidine incorporation in c.p.m. (mean ± S.D. of triplicated wells). Data are a representative of three independent experiments. ***, P, 0.001.

Fig 5

Cross-linking of HAb18G/CD147 with mAb 5A12 affects surface expression of CD25 and cytokine production. (A) mAb 5A12 down-regulates surface expression of CD25. Red-tinted histogram represents isotype control. Black and green lines represent anti-CD3 mAb plus isotype-matched irrelevant control mAb (mouse IgG1) or 5A12 intervention, respectively. (B, C, D) mAb 5A12 decreases IL-2 and IL-4 but increases IFN-γ production. Purified T cells were stimulated with immobilized 1 μg/ml of anti-CD3 mAb or 1 μg/ml of anti-CD3 mAb plus 10 μg/ml anti-CD28 mAb in the presence of 10 μg/ml mAb 5A12 or isotype-matched irrelevant control for 72 hrs. Bar graphs, (mean ± S.D.) levels of IL-2, IL-4 and IFN-γ in culture supernatants as measured by ELISA. *, P, 0.05.

Fig 6

Inhibition of anti-CD3 stimulated redistribution of the CD48 molecule by HAb18G/CD147 mAb 5A12. Purified peripheral blood T cells were cultured and stimulated with immobilized CD3 mAb in the presence or absence of 10 μg/ml of HAb18G/CD147 mAb 5A12 or isotype-matched irrelevant control mAb for 2 hrs. Then, cells were collected, fixed and stained with FITC-labelled CD48 as described in ‘Materials and methods’. The percentage of cells showing cap was assessed by analysing the distribution of the fluorescence intensity on the cell membrane of CD48-FITC stained cells using FV10-ASW (version 1.6) software. The images are a representative of three experiments. Scale bar, 5 μm. **, P, 0.01.

Fig 7

Cross-linking of HAb18G/CD147 with mAb 5A12 affects intracellular signalling pathway upon T cell activation. (A) Extracellular Ca²⁺ influx triggered by TCR stimulation is inhibited by mAb 5A12. The figure is a representative of three experiments. (B) HAb18G/CD147 mAb 5A12 slightly decreases the tyrosine phosphorylation level upon TCR triggering. Purified T cells were stimulated using CD3 mAb (plus mAb CD28) in the presence of mAb 5A12 or isotype-matched irrelevant control. After 2 hrs of stimulation, the cells were lysed and analysed for tyrosine phosphorylation level using Western blot. The figure is a representative of three experiments. Top, the representative image; bottom, quantitative analysis of the image.

Fig 8

Computer-assisted molecular docking of HAb18G/CD147 antigen-antibody complex. (A) The modelled structure of the variable domain of mAb HAb18 was docked to the most membrane-distal region of HAb18G/CD147 in a head-to-head manner. (B) The variable domain of mAb 6H8 was docked to C-terminal domain II of HAb18G/CD147. (C) The interaction between mAb 5A12 and HAb18G/CD147. The binding zone localizes at the functional N-terminal domain I. (D) Overview of docking the three mAbs to HAb18G/CD147 extracellular portion.