Fig 2: CD2/CD58 and LFA-1/ICAM-1 play important roles in TMP-mediated DC activation. a DCs (5 × 106) were treated with either PBS or 4-TMPs (5 × 107) for 2 h, and the transfer of indicated proteins on DCs (CD11c+) was analysed by flow cytometry. Data represent the mean of three experiments ± SEM. *P < 0.01 vs. no 4-TMPs. b Fluo-3-loaded DCs (5 × 106) were pretreated for 30 min with the indicated blocking antibodies (anti-CD2, -CD40, and -LFA-1), and then stimulated with 4-TMPs (5 × 107). Calcium signals were expressed as arbitrary units (a.u.) of Fluo-3 fluorescence from the average of at least three separate experiments. Data represent the mean of three experiments ± SEM. *P < 0.01 vs. PBS-treated group. c Representative images of cytokine arrays (left) and the heatmap showing cytokine-expression levels in total CD4+ T cell lysate and 4-TMPs (right). Each sample (200 µg) was hybridized to membranes containing capture antibodies specific for 112 cytokines. Data were quantified by densitometric analysis and statistically analysed. Cytokines overexpressed by up to 1.5-fold in TMPs relative to their expression in lysate are highlighted by red and blue boxes. Some cytokines were further confirmed by ELISA. Data represent the mean of three experiments ± SEM. *P < 0.01 vs. CD4+ T cell lysate

Fig 3: DOCK8+ CD4 T cell as T fh cell located at splenic red pulp, and its increase in peripheral blood of patients with SLE(A) a: Morphology of DOCK8+CD4 T cell observed by immunoelectron microscopy, stained with rabbit anti-DOCK8 Ab and Au-tagged ant-rabbit IgG Ab. (bar = 1µm; original magnification ×12,000). b,c: Nuclear membrane portions of DOCK8+ CD4 T cell, stained likewise. (bar = 1µm; original magnification ×18,000; x24,000).(B) Markers of splenic DOCK8+CD4 T cells. Flow cytometry for ICOS, CXCR5, and PD-1 expression in splenic DOCK8+CD4 T cells taken 9 days after the final immunization of BALB/c mice immunized 12x with PBS versus OVA (Left). Flow cytometry analysis of ICOS, CXCR5, PD-1, GATA3, ROR?t, T-bet, Bcl6, Ly6C, LFA1, FR4, and GL7 expression in splenic DOCK8-CD4 T cells and DOCK8+CD4 T cells isolated from 12x OVA-immunized BALB/c mice.(C) Cytokines released in culture supernatants of 1x106/ml DOCK8+ or DOCK8- CD4 T cells following incubation with 2 µg/mL of anti-CD3 and 5 µg/mL of anti-CD28 Ab at 37°C for 24hr. Data were represented as mean ± SEM. Statistical assessment was by Student's t-test. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001.(D) DOCK8+CD4 T cells in the autopsied spleen of untreated patient with active SLE who died before starting therapy. Tissues were fixed in 10% formalin, deparaffinized by graded xylene, and graded alcohol sequentially, and serially treated with biotinylated anti-DOCK8 monoclonal Ab and rabbit anti-IFN? Ab (for T cell staining), alkaline phosphatase avidin D, and anti-rabbit Novolink, followed by reaction with Fast Red (Red for DOCK8+ cells) and Histogreen (Blue for T cells). S: red pulp sinus. A; central artery. P: penicillar artery. (bar = 100µm; original magnification ×100). Inset; Magnified DOCK8+CD4 T cells (Arrows). (bar = 20µm; original magnification ×400).(E) Circulating DOCK8+CD4 T cells within CD4 T cells in the peripheral blood of patients with SLE and its relationship to the SLE disease activity index (SLEDAI). Data of control patients were depicted as; ¦ rheumatoid arthritis, ? mixed connective tissue disease, ? polymyositis, ? systemic sclerosis, ? microscopic polyangiitis. Also shown was quantification of interferona (IFNa) in sera of the same patients with SLE measured by ELISA.See also Figures S2 and S4–S6.