HBZ promotes proliferation of CD4⁺ T cells upon TCR stimulation.

<p>(A, B) Proliferation of CD4⁺ T cells isolated from non-Tg, tax-Tg and HBZ-Tg mice was analyzed by CFSE dilution. CD4⁺ T cells were stimulated with soluble anti-CD3 antibody (30 ng/mL) and cultured with or without dendritic cells for three days. (C) CD4⁺ T cells of non-Tg and HBZ-Tg mice were stimulated with immobilized anti-CD3 antibody (200 ng/mL) and soluble anti-CD28 antibody (0, 0.1, 0.3 and 1 μg/mL). CFSE dilution was analyzed by flow cytometry. (D) Experimental allergic encephalomyelitis (EAE) was induced in non-Tg and HBZ-Tg mice by immunization with MOG/CFA. The percentages of CD4⁺ T cells in spleen were measured in non-Tg and HBZ-Tg mice.</p

Interaction of HBZ with SHP associated factors.

<p>(A) Interaction between HBZ and THEMIS was analyzed by immunoprecipitation. (B) Interaction between THEMIS and, Grb2 or SHP-2, or HBZ was analyzed by immunoprecipitation. (A, B) Vectors expressing THEMIS, Grb2, SHP-2 and HBZ were transfected into 293FT cells (3.5×10⁶ cells, 10 cm dish). After 48 hours, transfected cells were stimulated with H₂O₂ for 5 min and cell lysates were immunoprecipitated with anti-PA antibody or normal rat IgG as a control. (C) THEMIS and Grb2 interaction in the presence or absence of HBZ was also analyzed with a densitometer. Relative protein levels were calculated as the ratio of immunoprecipitated protein to total protein.</p

Expression of TIGIT, PD-1, BTLA and Lair-1 in ATL cases.

<p>(A) Relative mRNA expression levels of co-inhibitory receptors in resting healthy donor CD4⁺ T cells (n = 5), PHA-activated CD4⁺ T cells (n = 5) and ATL cells (n = 14) were evaluated by real-time RT-PCR. (B) Cells from the same groups were stained with anti-CD4, TIGIT, PD-1, BTLA and LAIR-1 antibodies. Expression of co-inhibitory receptors on CD4⁺ T cells was analyzed by flow cytometry. (C) The MFI of TIGIT, PD-1, BTLA and LAIR-1 on the cells are shown.</p

Effect of HBZ on phosphorylation of SHP-2, ZAP-70, CD3ζ and PKCθ.

<p>(A) Phosphorylation levels of SHP-2 (Tyr580) in CD4⁺ T cells of non-Tg and HBZ-Tg mice were analyzed by flow cytometry. Splenocytes of non-Tg or HBZ-Tg mice (8 weeks old) were stained with anti-CD4 and pSHP-2 (Tyr580) antibodies. (B) The phosphorylation level of SHP-2 (Tyr580) of HBZ-transduced murine primary CD4⁺ T cells was analyzed by flow cytometry. After HBZ transduction, cells were stimulated with anti-CD3/PD-L1.Fc-coated beads at bead-to-cell ratio of 1:1 for 6 hours. (C-E) Dephosphorylation of ZAP-70, CD3ζ and PKCθ was analyzed by immunoblotting (left). Jurkat-mock and Jurkat-HBZ cells were stimulated with 20 mM H₂O₂ or 0.5 mM pervanadate for 0, 5, 15, 30 or 45 min. Phosphorylation levels were also analyzed by densitometry (right). Relative protein levels were calculated as the ratio of phosphorylated protein to total protein.</p

Schema of HBZ mediated inhibition of inhibitory signaling from co-inhibitory receptors.

<p>After T-cell activation through TCR, PD-1 forms microcluster with TCR complex. Complex containing SHP-2, Grb2 and THEMIS recruits to ITSM or ITIM motif of co-inhibitory receptors, PD-1 and TIGIT. SHP-1 dephosphorylates critical molecules for T-cell activation including ZAP-70 and CD3ζ. HBZ interacts with THEMIS, which changes subcellular localization of HBZ. HBZ interferes complex formation of THEMIS, Grb2 and SHP-2, which results in inhibition of suppressive signal from co-inhibitory receptors, and enhanced activation of T cells.</p

HBZ inhibits the suppressive effects of PD-1 and TIGIT.

<p>(A) HBZ-transduced murine primary CD4⁺ T cells were labeled with 5 μM CellTrace Violet and stimulated with anti-CD3/CD155.Fc-coated beads, anti-CD3/PD-L1.Fc-coated beads, or anti-CD3/control.Fc-coated beads at a bead-to-cell ratio of 1:1 for three days. CellTrace Violet dilution was analyzed by flow cytometry. (B) SHP-2 and PD-1 interaction in the presence or absence of HBZ was analyzed by immunoprecipitation. Vectors expressing SHP-2-Flag and HBZ (or mock)-IRES-mCD28/hPD1 were transfected into 293FT cells. After 48 hours, transfected cells were stimulated with pervanadate for 5 min and cell lysates were immunoprecipitated with anti-mCD28 antibody.</p

Expression of co-inhibitory receptors.

<p>(A) Transcripts of co-inhibitory and co-stimulatory receptor genes were quantified in HBZ transduced mouse T cells by real-time RT-PCR. (B) Splenocytes of non-Tg or HBZ-Tg mice (11 weeks old) were stained with anti-CD4, TIGIT, PD-1, BTLA and LAIR-1 antibodies. The expression of co-inhibitory receptors in CD4⁺ T cells was analyzed by flow cytometry. (C) The mean fluorescence intensity (MFI) of TIGIT, PD-1, BTLA and LAIR-1 in CD4⁺ T cells of non-Tg (n = 5) and HBZ-Tg (n = 5) mice is shown.</p

HBZ inhibits co-localization of PD-1 and SHP-2.

<p>(A) Staining of unstimulated or pervanadate-stimulated Jurkat-mock and Jurkat-HBZ cells was performed using antibodies against PD-1 (green) and SHP-2 (red). All scale bars are 5 μm. Three representative images derived from each sample are shown. Relative fluorescence intensities of PD-1 (green line) and SHP-2 (red line) were obtained on the white dotted line. (B) Co-localization of PD-1 and SHP-2 was judged by Pearson’s correlation coefficient between green and red channels. Each circle represents an individual cell. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test.</p