from spleen cells of 7 independent MRL+/+ and MRL/lpr mice. We found that the more responsive allele of P2X7R, the P451 isoform, is carried by the MRL genetic background and present in both MRL+/+ and MRL/lpr mice. Thus, an allelic variation cannot explain the differences in 
the levels of P2X7R signaling found between MRL+/+ and MRL/lpr mice. PMA/Iono Induces Shedding of CD62L in ATP-insensitive MRL/lpr T Cells Treatment of leukocytes with ATP or PMA is known to induce CD62L shedding via activation of the zinc-dependent membrane metalloprotease ADAM17 or TACE . However, PMA acts independently of the P2X7R pathway. Unlike ATP treatment, which activates CD62L shedding in T cells from MRL+/+, but not from MRL/lpr mice, PMA/Iono treatment induces CD62L shedding in T cells from both MRL+/+ and MRL/lpr mice. The shedding of CD62L by ATP in MRL+/+ T cells, or by PMA/Iono in MRL+/+ and 18519091 MRL/lpr T cells was prevented by the metalloprotease inhibitors GM6001 and Ro 31 9790, in a similar dose dependent-manner. These data show that while ADAM17 is present in T cells from both MRL+/+ and MRL/lpr mice, its activation by P2X7R signaling is dramatically impaired in MRL/lpr T cells, and also partially reduced upon PMA/Iono treatment. This reduced processing SCH 58261 price ATP-induced P2X7R Activation is Blocked in B220+ T Cells Only Unexpectedly, when comparing the responses of MRL/lpr and MRL+/+ spleen cells to ATP or NAD treatment measured by PS exposure and pore formation, we found that the Loss of P2X7R Expression on B220+ T Cells deficiency of P2X7R activity in MRL/lpr mice is age-related. Remarkably, the lack of ATP or NAD responsiveness in MRL/lpr mice can be reproducibly observed at later stages of the disease, but is not seen in younger MRL/lpr mice at earlier stages of the autoimmune syndrome. Since MRL+/+ spleen T cells respond to ATP or NAD treatment, the weaker response of spleen T cells from 4-mo-old MRL/lpr mice to stimulation by ATP or NAD is probably due to intrinsic change in the lymphocyte subsets i.e the appearance of high percentage of B220+ DN T cells in the spleen of MRL/lpr mice. The B220+ DN T-cell subset is present at a very low frequency in spleen and LNs from 1-mo-old MRL/lpr mice, and the frequency of this subset increases with age to become predominant in 3 to 4-mo-old MRL/lpr mice. Since As2O3 specifically eliminates B220+ DN T cells 20829789 in MRL/lpr mice as we had shown previously, we examined whether T cells from As2O3-treated MRL/lpr mice remained refractory to P2X7R stimulation, or whether they behaved like MRL+/+ T cells when challenged with ATP. In experiments starting at 2 months of age, MRL+/+ and MRL/lpr mice were treated with a daily injection of As2O3 or PBS for two months. As shown above, spleen cells from 4-mo-old PBS-treated MRL/lpr mice were resistant to 500 mM ATP. In contrast, spleen from 4-mo-old As2O3-treated MRL/lpr mice totally free of B220+ DN T cells responded to ATP treatment as efficiently as MRL+/+ T cells, indicating that the lack of responsiveness to stimulation by ATP or NAD observed in T cells from MRL/lpr mice at later stages of the disease is due to the accumulation of B220+ DN T cells with impaired P2X7R functions. During aging B220+ DN T cells accumulate in lpr LNs and spleen and dilute out conventional T cells. Thus, B220+ DN T cells represent 70.40611.55% and 26.264.25% of the T-cell population in 3 to 4-mo-old MRL/lpr mice and 5-mo-old B6/lpr mice, respectively. As expected, a small number of B220+ T cells w