In vivo dynamics of T cells and their interactions with dendritic cells in mouse cutaneous graft-versus-host disease.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation (alloSCT). By static microscopy, cutaneous GVHD lesions contain a mix of T cells and myeloid cells. We used 2-photon intravital microscopy to investigate the dynamics of CD4+ and CD8+ T cells and donor dendritic cells (DCs) in cutaneous GVHD lesions in an MHC-matched, multiple minor histocompatibility antigen-mismatched (miHA) model. The majority of CD4 and CD8 cells were stationary, and few cells entered and stopped or were stopped and left the imaged volumes. CD8 cells made TCR:MHCI-dependent interactions with CD11c+ cells, as measured by the durations that CD8 cells contacted MHCI+ vs MHCI- DCs. The acute deletion of Langerin+CD103+ DCs, which were relatively rare, did not affect CD8 cell motility and DC contact times, indicating that Langerin-CD103- DCs provide stop signals to CD8 cells. CD4 cells, in contrast, had similar contact durations with MHCII+ and MHCII- DCs. However, CD4 motility rapidly increased after the infusion of an MHCII-blocking antibody, indicating that TCR signaling actively suppressed CD4 movements. Many CD4 cells still were stationary after anti-MHCII antibody infusion, suggesting CD4 cell heterogeneity within the lesion. These data support a model of local GVHD maintenance within target tissues.

Donor-Specific Anti-HLA Antibodies in Allogeneic Hematopoietic Stem Cell Transplantation.

Allogeneic hematopoietic stem cell transplantation (AHSCT) is a curative treatment for a wide variety of hematological diseases. In 30% of the cases, a geno-identical donor is available. Any other situation displays some level of human leukocyte antigen (HLA) incompatibility between donor and recipient. Deleterious effects of anti-HLA immunization have long been recognized in solid organ transplant recipients. More recently, anti-HLA immunization was shown to increase the risk of primary graft failure (PGF), a severe complication of AHSCT that occurs in 3-4% of matched unrelated donor transplantation and up to 15% in cord blood transplantation and T-cell depleted haplo-identical stem cell transplantation. Rates of PGF in patients with DSA were reported to be between 24 and 83% with the highest rates in haplo-identical and cord blood transplantation recipients. This led to the recommendation of anti-HLA antibody screening to detect donor-specific antibodies (DSA) in recipients prior to AHSCT. In this review, we highlight the role of anti-HLA antibodies in AHSCT and the mechanisms that may lead to PGF in patients with DSA, and discuss current issues in the field.

Glutathione S-transferases interact with AMP-activated protein kinase: evidence for S-glutathionylation and activation in vitro.

AMP-activated protein kinase (AMPK) is a cellular and whole body energy sensor with manifold functions in regulating energy homeostasis, cell morphology and proliferation in health and disease. Here we apply multiple, complementary in vitro and in vivo interaction assays to identify several isoforms of glutathione S-transferase (GST) as direct AMPK binding partners: Pi-family member rat GSTP1 and Mu-family members rat GSTM1, as well as Schistosoma japonicum GST. GST/AMPK interaction is direct and involves the N-terminal domain of the AMPK ß-subunit. Complex formation of the mammalian GSTP1 and -M1 with AMPK leads to their enzymatic activation and in turn facilitates glutathionylation and activation of AMPK in vitro. GST-facilitated S-glutathionylation of AMPK may be involved in rapid, full activation of the kinase under mildly oxidative physiological conditions.

A two-dimensional screen for AMPK substrates identifies tumor suppressor fumarate hydratase as a preferential AMPKα2 substrate.

AMP-activated protein kinase (AMPK) is emerging as a central cellular signaling hub involved in energy homeostasis and proliferation. The kinase is considered as a suitable target for pharmacological intervention in several energy-related pathologies like diabetes type II and cancer, although its signaling network is still incompletely understood. Here we apply an original two-dimensional in vitro screening approach for AMPK substrates that combines biophysical interaction based on surface plasmon resonance with in vitro phosphorylation. By enriching for proteins that interact with a specific AMPK isoform, we aimed to identify substrates that are also preferentially phosphorylated by this specific AMPK isoform. Application of this screen to full-length AMPK α2ß2γ1 and soluble rat liver proteins identified the tumor suppressor fumarate hydratase (FH). FH was confirmed to interact with and to be preferentially phosphorylated by the AMPKα2 isoform by using yeast-two-hybrid and in vitro phosphorylation assays. AMPK-mediated phosphorylation of FH significantly increased enzyme activity in vitro and in vivo, suggesting that it is a bona fide AMPK substrate. In vivo, AMPKα2 is supposed to target the cytosolic/nuclear pools of FH, whose tumor suppressor function relies on DNA damage repair and inhibition of HIF-1α-signaling.