Expression and function of CXCR3 on CD4⁺ T cells in HBZ-Tg and HBZ-Tg/IFN-γ KO mice.

<p>(A) Splenocytes were obtained from mice, and the CXCR3 expression level in CD4⁺ T cells was evaluated by flow cytometry. Three mice of each strain were analyzed and the result was summarized in the graph. (B) The migration activity of CD4⁺ T cells towards CXCL10. CD4⁺ T cells were isolated from splenocytes using magnet beads. Murine recombinant CXCL10 was added at concentrations of 0, 200, or 500 ng/mL. Migrating cells were counted by flow cytometry.</p

CXCL10 is not associated with systemic inflammation in HBZ-Tg mice.

<p>(A) HBZ-Tg and CXCL10 KO mice were crossed to establish HBZ-Tg/CXCL10 KO mice. (B) HBZ-Tg/CXCL10 KO mice developed dermatitis. (C) At 24 weeks old, more than 50% of HBZ-Tg/CXCL10 KO mice developed dermatitis. (D) Splenocytes were obtained from CXCL10 KO mice and HBZ-Tg/CXCL10 KO mice. Cells were stained with anti-CD4, anti-CD44, anti-CD62L, and anti-Foxp3, and analyzed by flow cytometry. Among CD4⁺ T cells of HBZ-Tg/CXCL10 KO mice there were increased numbers of increased effector/memory and Foxp3 expressing cells.</p

Incidence of inflammation and lymphoma is decreased in HBZ-Tg/IFN-γ KO mice.

<p>(A) HBZ-Tg and HBZ-Tg/IFN-γ KO mice at 24 weeks of age. HBZ-Tg mice developed dermatitis around the ears, eyes, and back. HBZ-Tg/IFN-γ KO mice did not develop dermatitis. (B) 90% of HBZ-Tg developed dermatitis by 2 years of age. However, in HBZ-Tg/IFN-γ KO mice, the onset of dermatitis was delayed and its incidence was lower than in HBZ-Tg mice. *** indicates significant differences p<0.01. (C) Histological analysis of HBZ-Tg and HBZ-Tg/IFN-γ KO mice (x10). The spleen of an HBZ-Tg mouse shows a diminution of the structure and a diffuse infiltration of atypical cells in red palps. The lymph node of HBZ-Tg shows a focal diminution of nodal structures and a diffuse infiltration of atypical cells in paracortical area. The skin of HBZ-Tg shows hyperkeratosis of epidermis. However, the spleen, lymph node and skin of HBZ-Tg/IFN-γ KO show normal structures. (D) Lymphomas developed in spleen and lymph nodes of HBZ-Tg mice with dermatitis. In the high magnification (x60), the spleen of an HBZ-Tg mouse shows a diffuse infiltration of atypical large lymphoid cells with irregular nuclei, and the lymph node shows atypical large lymphoid cells, also. Abnormal cells are indicated by white arrows.</p

Histological findings in CXCL10 KO, and HBZ-Tg/CXCL10 KO mice at 24 weeks of age.

<p>Histological findings in CXCL10 KO, and HBZ-Tg/CXCL10 KO mice at 24 weeks of age.</p

Microarray analysis of WT, HBZ-Tg, IFN-γ KO, and HBZ-Tg/IFN-γ KO mice.

<p>CD4⁺ T cells were purified from splenocytes of WT, HBZ-Tg, IFN-γ KO, and HBZ-Tg/IFN-γ KO mice. (A) Validation of the microarray result by qPCR. cDNA of splenocytes from WT, HBZ-Tg, and HBZ-Tg/IFN-γ KO mice were used. Expression levels of the candidate genes were normalized using the values of WT as reference. (B) Transcription levels of the human homologues of the candidate genes in ATL patients and healthy donors (HD). Relative expression values were calculated by the delta delta Ct method using a value of one resting sample as reference. Aggressive: acute and lymphoma types of ATL.</p

Inflammatory phenotypes of germ-free HBZ-Tg mice.

<p>(A) GF HBZ-Tg mice developed dermatitis similarly to SPF HBZ-Tg mice. (B) Splenocytes were harvested from 18-week-old GF HBZ-Tg or GF WT littermates. The percentages of Tregs and effector/memory CD4⁺ T cells were evaluated. Representative results of the dot plots and a summarized table are shown. (C) Cytokine production in CD4⁺ T cells was evaluated. Splenocytes were stimulated with PMA/ionomycin in the presence of protein transport inhibitor for 4 hours, stained with specific antibodies, and analyzed by flow cytometry. Representative results of the dot plots and a summarized table are shown.</p

Histological findings in WT, HBZ-Tg, IFN-γ KO, and HBZ-Tg/IFN-γ KO mice at 24 weeks of age.

<p>Histological findings in WT, HBZ-Tg, IFN-γ KO, and HBZ-Tg/IFN-γ KO mice at 24 weeks of age.</p

Comparison of T-cell subsets between HBZ-Tg and HBZ-Tg/IFN-γ KO mice.

<p>(A and B) Splenocytes were harvested from WT, HBZ-Tg, IFN-γ KO, and HBZ-Tg/IFN-γ KO mice at 24-week old. Cells were stained with anti-CD4, anti-Foxp3, anti-CD25 antibodies for detection of regulatory T cells, and anti-CD44, anti-CD62L antibodies for effector/memory CD4⁺ T cells. Percentages of each subset in CD4⁺ T cells are shown (n = 3 or 4). TN: naïve T cell, TEM: effector/memory T cell. (C and D) Cytokine production in CD4⁺Foxp3^- T cells of each strain was evaluated. Splenocytes were stimulated with PMA/ionomycin in the presence of protein transport inhibitor for 4 hours, stained with specific antibodies, and analyzed by flow cytometry. Percentages of each subset in CD4⁺ T cells are shown (n = 3 or 4).</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

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