B6.CCR5?/? mice were managed in the Lerner Research Institute Biological Resources Unit. surface antigen NK1.1, with NK1.1low cells reflecting the highest level of activation. These NK cell populations increased with time post-transplant. In contrast, NK cell infiltration into semi-allogeneic grafts on day 7 was composed entirely of NK1.1high cells that decreased thereafter. On day 65 post-transplant the semi-allogeneic grafts experienced severe interstitial fibrosis, glomerulopathy, and arteriopathy, accompanied by expression of pro-fibrogenic genes. These results suggest that NK cells synergize with DSA to cause acute kidney allograft rejection, whereas high DSA titers in the absence of NK cell activation cannot provoke acute ABMR but instead induce the indolent development of interstitial fibrosis and glomerular injury that leads to late graft failure. 0.05, ** 0.01, and *** 0.001. NK cells express PD153035 (HCl salt) surface activating and inhibitory receptors and engagement of self class I MHC transduces dominant inhibitory signals that attenuate NK cell activation (20, 21). Therefore, we postulated that expression of recipient class I MHC with allogeneic class I MHC on kidney grafts would prevent NK cell activation during ABMR. We crossed the allogeneic donor A/J and recipient background C57BL/6 mice to generate (A/J x B6)F1 semi-allogeneic graft donors for B6.CCR5?/? recipients and NK cell infiltration and activity in the F1 semi-allogeneic kidney grafts was examined. NK cell infiltration into F1 kidney grafts was observed on day 7 post-transplant at numbers equivalent to those observed infiltrating complete MHC-mismatched A/J kidney allografts. In contrast to the NK cells infiltrating the A/J allografts, all NK cells infiltrating the semi-allogeneic F1 PD153035 (HCl salt) grafts on day 7 post-transplant were entirely NK1.1high cells with few NK1.1low cells (Figure 2A-C) and very few expressed CD107a (Figure 2D-F). Moreover, NK cell numbers in F1 grafts fell to near baseline/isograft numbers by day 14 post-transplant. Overall these results indicated increasing infiltration, accumulation and activation of NK cells into complete MHC-mismatched kidney allografts and early post-transplant infiltration of NK cells into semi-allogenic grafts that decreased after day 7 post-transplant. NK cell proliferation in complete MHC mismatched A/J allografts Since NK1.1high and NK1.1low cells increased over time post-transplant in A/J allografts, we tested their proliferation within the allografts using BrdU labeling (Figure 3A). The total number of BrdU+ NK cells increased from day 7 to 14 post-transplant (Figure 3B), with no apparent difference in the percentage of proliferating NK1.1high and NK1.1low PD153035 (HCl salt) cells (Figure 3C). While the number of BrdU+ NK1. 1low cells within allografts was significantly lower than that of BrdU+NK1.1high cells on day 7 post-transplant, these increased to equivalent numbers as BrdU+NK1.1high cells by day 14 (Figure 3 D). These results suggested that the increased number of NK1.1low cells from day 7 to 14 post-transplant was due, at least in part, to the proliferation of NK1.1low cells within the allograft. Open in a separate window Figure 3 I Proliferation of NK cells infiltrating complete MHC- mismatched A/J kidney allografts.Groups of B6.CCR5?/? mice Rabbit polyclonal to ZNF76.ZNF76, also known as ZNF523 or Zfp523, is a transcriptional repressor expressed in the testis. Itis the human homolog of the Xenopus Staf protein (selenocysteine tRNA genetranscription-activating factor) known to regulate the genes encoding small nuclear RNA andselenocysteine tRNA. ZNF76 localizes to the nucleus and exerts an inhibitory function onp53-mediated transactivation. ZNF76 specifically targets TFIID (TATA-binding protein). Theinteraction with TFIID occurs through both its N and C termini. The transcriptional repressionactivity of ZNF76 is predominantly regulated by lysine modifications, acetylation and sumoylation.ZNF76 is sumoylated by PIAS 1 and is acetylated by p300. Acetylation leads to the loss ofsumoylation and a weakened TFIID interaction. ZNF76 can be deacetylated by HDAC1. In additionto lysine modifications, ZNF76 activity is also controlled by splice variants. Two isoforms exist dueto alternative splicing. These isoforms vary in their ability to interact with TFIID received complete PD153035 (HCl salt) MHC-mismatched A/J allografts (n = 6 grafts per time point). BrdU was injected i.p. on day 6 or day 13 post-transplant and grafts were harvested and weighed the following day on day 7 or 14 post-transplant. Each graft was digested to obtain single cell suspensions and aliquots were stained with fluorochrome-labeled mAb for flow cytometry analysis of total NK cells and the NK1.1high and NK1.1low cell populations in each graft. (A) Representative gating of BrdU+NK cells in flow cytometry analysis of allograft infiltrating NK1.1high and NK1.1low cell populations on days 7 and 14 post-transplant. (B) Absolute number of total BrdU+NK cells/mg graft tissue on day 7 and 14 post-transplant. (C) The frequency of BrdU+ NK1.1high and NK1.1low cell populations infiltrating allografts on days 7 and 14 post-transplant. (D) The numbers of allograft infiltrating BrdU+ NK1.1high and NK1.1low cells/mg PD153035 (HCl salt) graft tissue on days 7 and 14 post-transplant. *p 0.05, **p 0.01. NK1.1.