The tetraspanin CD9 is preferentially expressed on the human CD4(+)CD45RA+ naive T cell population and is involved in T cell activation

Abstract

Human CD4+ T cells can be divided into reciprocal memory and naive T cell subsets based on their expression of CD45 isoforms and CD29/integrin beta1 subunit. To identify unique cell surface molecules on human T cells, we developed a new monoclonal antibody termed anti5H9. Binding of anti5H9 triggers a co-stimulatory response in human peripheral blood T cells. Retrovirus-mediated expression cloning has revealed that the antigen recognized by anti5H9 is identical to the tetraspanin CD9. We now show that human CD9 is preferentially expressed on the CD4(+)CD45RA+ naive T cell subset, and that CD9(+)CD45RA+ T cells respond preferentially to the recombinant beta2-glycoprotein I, compared to CD9-CD45RA+ T cells. Furthermore, anti5H9 inhibits both the recombinant beta2-glycoprotein I- and the recall antigen tetanus toxoid-specific T cell proliferation. These results suggest that the tetraspanin CD9 plays an important role in T cell activation.

Fig. 1

5H9 antigen is identified to be the CD9 molecule. (a) Reactivity of anti5H9 MoAb to human peripheral blood T cells. Two-colour staining analysis of freshly isolated human PBMC was performed using FITC-conjugated anti5H9, RD1-conjugated anti-CD3. Numbers indicate the relative percentages of positive cells within a quadrant. The result is representative of five separate experiments. (b) Reactivity of anti-CD9 MoAbs on H9 cells transfected with isolated cDNA of the 5H9 antigen. H9 parent cells as a negative control (a–d) or H9–5H9-1 cells transfected with isolated cDNA of 5H9 antigen (e–h) were stained with mouse IgG (a and e), anti5H9 (b and f), ALB6 (anti-CD9) (c and g), 4B4 (anti-CD29) (d and h) and FITC-conjugated goat antimouse IgG. (c) Western blotting of 5H9 antigen. H9–5H9-1 cells were lysed in lysis buffer. The lysates were separated on 12% SDS-PAGE under reducing (R) or non-reducing (NR) condition, and then immunoblotting was carried out with anti5H9 (blot). The positions of molecular weight markers are indicated on the left (MW). (d) Co-stimulatory effect of anti5H9 with the immobilized submitogenic dose of anti-CD3 on human peripheral blood T cells. Left: dose–response curve; right: time–course curve. Purified T cells at 2·0 × 10⁵ cells/well were stimulated with immobilized suboptimal concentration of anti-CD3 (OKT3) and the indicated concentrations of anti5H9 or anti-CD28 (4B10). Proliferation was assessed by [³H]-thymidine (1 µCi/well) incorporation assay in the last 13 h of cultures. The result is representative of three independent experiments and expressed as mean cpm ± s.d. of triplicate cultures.

Fig. 2

Preferential expression of CD9 on CD4⁺CD45RA⁺ human naive T cells. CD9 defined by anti5H9 is present on a subpopulation of human peripheral blood T cells. Four-colour staining analysis of freshly isolated human PBMC was performed using FITC-conjugated anti-CD45RO, PE-conjugated anti-CD45RA, PerCP-conjugated anti-CD4, biotinylated anti5H9 and APC-conjugated streptavidin (a) and FITC-conjugated anti-CD45RA, PerCP-conjugated anti-CD4, PE-conjugated anti-CD62L, biotinylated anti5H9 and APC-conjugated streptavidin (b). Numbers indicate the relative percentages of positive cells within a quadrant. The result is representative of five separate experiments.

Fig. 3

Differential response of CD45RA⁺ and CD45RO⁺ T cells to recombinant beta₂-GPI and tetanus toxoid (TT). (a) CD45RA⁺ T cells and CD45RO⁺ T cells were enriched by negative selection, and their purity was analysed by flow cytometry; unfractionated: pre-enrichment, CD45RA enriched: CD45RA⁺ T cells were enriched (>95% positive), CD45RO enriched: CD45RO⁺ T cells were enriched (>95% positive). (b) The proliferative response to beta₂-GPI or TT was assessed with incorporation of [³H]-thymidine. Media alone (–), beta₂-GPI (5 µg/ml), TT (5 ng/ml) and PHA (5 µg/ml) were used. The data are expressed as mean cpm ± s.d. of triplicate samples. Representative data of three separate experiments are shown. (c) Comparison between proliferative response of CD9⁺CD45RA⁺ T cells and CD9^–CD45RA⁺ T cells to beta₂-GPI (5 µg/ml), TT (5 ng/ml) and PHA (5 µg/ml) by T cell proliferation assay; unfractionated (unfractionated T cells population), CD45RA⁺ pre-sort (a population without anti5H9 treatment for cell sorting), and CD45RA⁺ (a population with anti5H9 treatment for cell sorting) T cells were also examined. The data are expressed as mean cpm ± s.d. of sextuple samples. Representative data of three separate experiments are shown.

Fig. 4

Effect of anti5H9 on T cell proliferation induced by recombinant beta₂-GPI and TT. (a) The effect on proliferative response to recombinant beta₂-GPI (5 µg/ml) (left panel) and TT (5 ng/ml) (right panel) was assessed with various concentrations of MoAb, isotype-matched control MoAb (anti4B4), anti5H9 and anti-HLA DR MoAb (anti-L243). Data are expressed as mean cpm ± s.d. of triplicate samples. Representative data of three independent experiments are shown. *Significant inhibition compared to the culture with isotype-matched control MoAb (P < 0·05). (b) Inhibitory effect of anti5H9 (15 µg/ml) on beta₂-GPI-induced T cell proliferation was compared with those of various MoAbs (15 µg/ml). Data are expressed as mean cpm ± s.d. of sextuple samples. Representative data of three independent experiments are shown. *Significant inhibition compared to the culture without MoAb (P < 0·05).