High doses of Nec-1 inhibit T cell proliferation.

Abstract

<p>(A) Low dose Nec-1 inhibits TCR-induced necrosis, but high dose Nec-1 inhibits T cell proliferation. Purified T cells from wild type or FADD^−/− mice were stimulated with plate-bound anti-CD3, anti-CD28 and increasing doses of Nec-1. Three days later, cell proliferation was measured by incorporation of [³H]-thymidine. Results shown are mean ± SEM. (B) Nec-1 did not compromised T cell viability. Naïve CD3⁺ T cells were purified from the spleen of wild type C57BL/6 mice and incubated at 37°C for 24 hours in the presence or absence of 50 µM Nec-1. Viability of the cells was determined by flow cytometry using propidium iodide (PI) uptake as an indication of cell death. Note that Nec-1 increased the baseline fluorescence of the T cells. (C) Nec-1 inhibits T cell division. Purified CD3⁺ primary T cells were labeled with CellTracer Violet fluorescent dye and stimulated with 1 µg/ml plate-bound anti-CD3 and 200 ng/ml anti-CD28 antibodies. Three days later, cell division was analyzed using a BD LSR2 flow cytometer. The numbers above each peak represent the number of cell division the cells had undergone. The numbers on the left represent the percentages of cells in each peak. (D) Nec-1 inhibits T cell blast formation. Purified CD3⁺ T cells were similarly activated as in (C). Three days later, formation of T cell blast as measured by forward scatter was determined by flow cytometry. (E) FADD^−/−RIP1^−/− DKO T cells stimulated with plate-bound anti-CD3 and anti-CD28 antibodies in the absence or presence of Nec-1 were measured for cell proliferation as in (C).</p