Uc10 4f10 11

The retroviral supernatants containing the CTLA4-CD28 chimera retrovirus and the empty-vector retrovirus were generated as previously described.^{36 (link)} The retroviruses also encoded the IRES-GFP module to enable the tracing of the transduced cells (pMIG-w). For the retroviral transduction of T cells, spleen and lymph node cells were isolated from B10.A mice and stimulated with plate-bound anti-CD3 (10 μg ml⁻¹, 145-2C11) and anti-CD28 (2 μg ml⁻¹, 37.51) antibodies (BD Biosciences). After 24 h, the T cells were transduced with the concentrated retroviruses by centrifugation of the cells at 2500 r.p.m. for 90 min (spin infection). This procedure was repeated once on the same day. During the spin infection, 6 μg ml⁻¹ Polybrene (Sigma-Aldrich, St Louis, MO, USA) was added to the culture supernatant to enhance the transduction efficiency. After 48 h, the transduced T cells were transferred into fresh medium containing 20 U ml⁻¹ recombinant mouse IL-2 (Invitrogen, Waltham, MA, USA) and allowed to rest for 72 h without stimulation. After resting, more than 95% of the live cells were CD4 or CD8 T cells. The cells were stained with phycoerythrin (PE)-conjugated anti-CTLA4 antibody (UC10-4F10-11, BD Biosciences) and analyzed by flow cytometry to measure the expression of GFP and cell surface CTC28.