Down-regulation of IEX-1 expression following T cell activation. PBT cells were stimulated with anti-CD3 Ab and collected at 2 h (day 0) or indicated days. The cells were either left unstimulated (day 0) or restimulated with anti-CD3 Ab for another 2 h, and stained with FITC-anti-CD2 Ab and phycoerythrin (PE)-conjugated-anti-Fas Ab (Left) or anti-IEX-1 specific polyclonal Ab (Right). Aliquots of cells were treated with anti-Fas Ab in the presence of 100 units/ml of IL-2. The percentages of apoptotic cell death were determined on a gated CD2⁺ T cell population and indicated next to the FACS panels on the right. One representative result of two (Fas expression and Fas-induced apoptosis) and four (IEX-1 expression) experiments performed is shown.

Splenomegaly, lymphadenopathy, and accumulation of effector/memory-like T lymphocytes in IEX-1-Tg mice. (A) RT-PCR analysis of IEX-1 mRNA in IEX-1-Tg mice. Total RNA was isolated from LN T cells of non-Tg mice (lane 1), and IEX-1-Tg mice Tg-2 (lane 2) and Tg-4 (lane3), derived from founders 2 and 4, respectively, and assayed by RT-PCR using primers that amplified human IEX-1 only. Lower is amplification of G3PDH (glyceraldhyde-3-phosphate dehydrogenase) from the same RT-reaction samples for controls. (B) Relative IEX-1 protein expression in IEX-1-Tg mice. LN T cells isolated from above mice were labeled with FITC-conjugated anti-CD3 Ab followed by staining with anti-IEX-1 polyclonal Ab (solid) or preimmunized normal rabbit Ab (outlined) as in Fig. 1. The mean immunofluorescence values of anti-IEX-1 Ab staining on gated CD3⁺ cells are indicated in each profile. (C) Splenomegaly and lymphadenopathy in IEX-1-Tg mice. Shown are the spleen and LN (superficial inguinal) from 9–10-month-old IEX-1-Tg mice (b, d, e, and f) and non-Tg age-matched control mice (a and c). (D) Northern blotting analysis of IEX-1 expression in T and B cells. Trangenic or human IEX-1 (hIEX-1) and endogenous mouse IEX-1 (mIEX-1) expressed in B (lane 1) and T (lane 2) cells are indicated by arrows on the right of the upper panel. T cells from non-Tg mice (T-) expressed mIEX-1 only following stimulation with ti-CD3 Ab for 2 h as a positive control (lane 3). RNA loading control is given in the low panel. (E) A comparison of splenocyte phenotypes between IEX-1-Tg (Tg) and non-Tg (Non-Tg) mice. Splenocytes were stained with indicated Abs and analyzed by three-colored flow cytometry. One representative result of eight IEX-1-Tg and six non-Tg mice analyzed is shown; the data obtained from all mice are summarized in Table 1.

Impaired apoptosis in T cells from IEX-1-Tg mice. T cells from IEX-1-Tg (Tg) and non-Tg (Non-Tg) littermates at 6–8 weeks of age underwent apoptosis triggered by anti-CD3 Ab (A) or anti-Fas Ab (B) following activation. (A) The means from triplicate samples of each mouse with a total of ten IEX-1-Tg and eight non-Tg mice performed. Standard deviation (SD) of the triplicate cultures was less than 3% (data not shown) and each symbol represents an individual mouse. (B) The means ± SD of Fas-induced apoptosis in T cells (n = 5), which represents one result of three experiments performed in triplicate. (C) A defect in superantigen-mediated depletion of T cells in IEX-1-Tg mice. IEX-1-Tg and non-Tg littermates were administrated with 30 μg of SEB. Percentages of CD4⁺Vβ8⁺ T cells relative to total CD4⁺ T cells in the spleens were determined at the indicated days by flow cytometric analysis. Data are shown as means (n = 6) ± SD of three independent experiments in which two mice per time point were used for each experiment.

An elevated and extended DTH response in IEX-1-Tg mice. A DTH response was induced in IEX-1-Tg (Tg) mice and non-Tg (Non-Tg) littermates by intracutaneous injection of Mycobacterium tuberculosis into the right hind footpad (Left). The mice were immunized again with the same antigen in the left hind footpads one-month later (Right). The footpad thickness (mm) was measured daily by a caliper. Dada are shown as means (n = 7) ± SD of footpad thickness. One representative result of three experiments performed is shown.

A lupus-like autoimmune disease in IEX-1-Tg mice. (A and B) Hematoxylin and eosin (H&E)-stained kidney sections: a glomerulus from a 9-month-old non-Tg mouse and an age-matched IEX-1-Tg mouse, respectively. (C and D) Immunofluorescent staining of kidney sections with FITC-conjugated goat anti-mouse IgG Ab. Note that fluorescence in glomeruli of a 9-month-old IEX-1-Tg mouse (D), but not in glomeruli of a non-Tg age-matched mouse (C). (E and F) Erythema with alopecia in the skin (E) and swelling of joint (F, a) in IEX-1-Tg mice; b in F, normal foot control.