FoxP3+, and not CD25+, T cells increase post-transplant in islet allotransplant recipients following anti-CD25+ rATG immunotherapy.

Anti-CD25 antibodies are used as an induction therapy in islet allotransplantation for type 1 diabetes. Although previous reports suggested that anti-CD25 treatment may lead to depletion of CD4+CD25+ regulatory T cells (Tregs) and questioned its use in tolerance-promoting protocols for transplantation, the effect of anti-CD25 antibodies on the frequency and function of Tregs remains unclear. We examined the effect of anti-CD25 antibody, daclizumab, in vivo on Tregs in islet allograft recipients enrolled in a single-center study and monitored post-transplant. Our data shows that the reduction in CD25+ Treg cells observed post-transplant is due to masking of CD25 receptor by daclizumab and not due to depletion. In addition, using Treg marker, FoxP3, we show that anti-CD25+ ATG treatment leads to an increase in FoxP3+ Tregs post-transplant. These data suggest that anti-CD25-based therapy has beneficial effects on Tregs and combined with ATG may be a promising therapy for autoimmunity and transplantation.

C-terminal domain modulates the nucleic acid chaperone activity of human T-cell leukemia virus type 1 nucleocapsid protein via an electrostatic mechanism.

Retroviral nucleocapsid (NC) proteins are molecular chaperones that facilitate nucleic acid (NA) remodeling events critical in viral replication processes such as reverse transcription. Surprisingly, the NC protein from human T-cell leukemia virus type 1 (HTLV-1) is an extremely poor NA chaperone. Using bulk and single molecule methods, we find that removal of the anionic C-terminal domain (CTD) of HTLV-1 NC results in a protein with chaperone properties comparable with that of other retroviral NCs. Increasing the ionic strength of the solution also improves the chaperone activity of full-length HTLV-1 NC. To determine how the CTD negatively modulates the chaperone activity of HTLV-1 NC, we quantified the thermodynamics and kinetics of wild-type and mutant HTLV-1 NC/NA interactions. The wild-type protein exhibits very slow dissociation kinetics, and removal of the CTD or mutations that eliminate acidic residues dramatically increase the protein/DNA interaction kinetics. Taken together, these results suggest that the anionic CTD interacts with the cationic N-terminal domain intramolecularly when HTLV-1 NC is not bound to nucleic acids, and similar interactions occur between neighboring molecules when NC is NA-bound. The intramolecular N-terminal domain-CTD attraction slows down the association of the HTLV-1 NC with NA, whereas the intermolecular interaction leads to multimerization of HTLV-1 NC on the NA. The latter inhibits both NA/NC aggregation and rapid protein dissociation from single-stranded DNA. These features make HTLV-1 NC a poor NA chaperone, despite its robust duplex destabilizing capability.

Retroviral nucleocapsid proteins display nonequivalent levels of nucleic acid chaperone activity.

Human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) is a nucleic acid chaperone that facilitates the remodeling of nucleic acids during various steps of the viral life cycle. Two main features of NC's chaperone activity are its abilities to aggregate and to destabilize nucleic acids. These functions are associated with NC's highly basic character and with its zinc finger domains, respectively. While the chaperone activity of HIV-1 NC has been extensively studied, less is known about the chaperone activities of other retroviral NCs. In this work, complementary experimental approaches were used to characterize and compare the chaperone activities of NC proteins from four different retroviruses: HIV-1, Moloney murine leukemia virus (MLV), Rous sarcoma virus (RSV), and human T-cell lymphotropic virus type 1 (HTLV-1). The different NCs exhibited significant differences in their overall chaperone activities, as demonstrated by gel shift annealing assays, decreasing in the order HIV-1 approximately RSV > MLV >> HTLV-1. In addition, whereas HIV-1, RSV, and MLV NCs are effective aggregating agents, HTLV-1 NC, which exhibits poor overall chaperone activity, is unable to aggregate nucleic acids. Measurements of equilibrium binding to single- and double-stranded oligonucleotides suggested that all four NC proteins have moderate duplex destabilization capabilities. Single-molecule DNA-stretching studies revealed striking differences in the kinetics of nucleic acid dissociation between the NC proteins, showing excellent correlation between nucleic acid dissociation kinetics and overall chaperone activity.

Potentially immunogenic proteins expressed similarly in human embryonic stem cells and induced pluripotent stem cells.

A major limitation of the use of cellular therapies is the loss of donor-derived cells because of immune incompatibility. While induced pluripotent stem (iPS) cells offer the potential for autologous transplant therapies, questions have been raised using a mouse model that specific antigens mediate the rejection of grafts after syngeneic transplants with iPS, but not embryonic stem (ES) cells. In this study, we examined whether the human homologs of these markers, HORMAD1, ZG16, and Cyp3A, are differentially expressed in human iPS versus ES cells, as well as undifferentiated and in vitro-differentiated cells. Both qRT-PCR and flow cytometric analyses demonstrated similar gene and protein expression profiles for iPS and ES cells regardless of differentiation state. Our data are consistent with a recent study in mice that showed no evidence of rejection of differentiated syngeneic iPS cells. Furthermore, our results suggest that expression of these gene products cannot predict differences in clinical outcomes between human iPS and ES-derived cells.