Inactivation of human immunodeficiency virus (HIV)-1 envelope-specific CD8+ cytotoxic T lymphocytes by free antigenic peptide: A self-veto mechanism?

Free peptide has been found to inhibit cytotoxic T lymphocyte (CTL) activity, and veto cells bearing peptide-major histocompatibility complex (MHC) complexes have been found to inactivate CTL, but the two phenomenon have not been connected. Here we show that a common mechanism may apply to both CD8+ CTL lines or clones specific for a determinant of the human immunodeficiency virus (HIV)-1 IIIB envelope protein gp160, P18IIIB, are inhibited by as little as 10 min exposure to the minimal 10-mer peptide, 1- 10, within P18IIIB, free in solution, in contrast to peptide already bound to antigen-presenting cells (APC), which does not inhibit. Several lines of evidence suggest that the peptide must be processed and presented by H-2D(d) on the CTL itself to the specific T cell receptor (TCR) to be inhibitory. The inhibition was not killing, in that CTL, did not kill 51Cr-labeled sister CTL in the presence of free peptide, and in missing experiments with CTL lines of different specificities restricted by the same MHC molecule, D(d), the presence of free peptide recognized by one CTL line did not inhibit the activity of the other CTL, line that could present the peptide. Also, partial recovery of activity could be elicited by restimulation with cell-bound peptide, supporting the conclusion that neither fratricide nor suicide (apoptosis) was involved. The classic veto phenomenon was ruled out by failure of peptide-bearing CTL to inactivate others. Using pairs of CTL lines of differing specificity but similar MHC restriction, each pulsed with the peptide for which the other is specific, we showed that the minimal requirement is simultaneous engagement of the TCR and class I MHC molecules of the same cell. This could occur in single cells or pairs of cells presenting peptide to each other. Thus, mechanistically the inhibition is analogous to veto, and might be called self-veto. As a clue to a possible mechanism, we found that free 1-10 peptide induced apparent downregulation of expression of specific TCR as well as interleukin 2 receptor, CD69, lymphocyte function-associated antigen 1, and CD8. This self-veto effect also has implication for in vivo immunization and mechanisms of viral escape from CTL immunity

MRI Tracking of FePro Labeled Fresh and Cryopreserved Long Term In Vitro Expanded Human Cord Blood AC133+ Endothelial Progenitor Cells in Rat Glioma

Background: Endothelial progenitors cells (EPCs) are important for the development of cell therapies for various diseases. However, the major obstacles in developing such therapies are low quantities of EPCs that can be generated from the patient and the lack of adequate non-invasive imaging approach for in vivo monitoring of transplanted cells. The objective of this project was to determine the ability of cord blood (CB) AC133+ EPCs to differentiate, in vitro and in vivo, toward mature endothelial cells (ECs) after long term in vitro expansion and cryopreservation and to use magnetic resonance imaging (MRI) to assess the in vivo migratory potential of ex vivo expanded and cryopreserved CB AC133+ EPCs in an orthotopic glioma rat model. Materials, Methods and Results: The primary CB AC133+ EPC culture contained mainly EPCs and long term in vitro conditions facilitated the maintenance of these cells in a state of commitment toward endothelial lineage. At days 15–20 and 25–30 of the primary culture, the cells were labeled with FePro and cryopreserved for a few weeks. Cryopreserved cells were thawed and in vitro differentiated or IV administered to glioma bearing rats. Different groups of rats also received long-term cultured, magnetically labeled fresh EPCs and both groups of animals underwent MRI 7 days after IV administration of EPCs. Fluorescent microscopy showed that in vitro differentiation of EPCs was not affected by FePro labeling and cryopreservation. MRI analysis demonstrated that in vivo accumulation of previously cryopreserved transplanted cells resulted in significantly higher R2 and R2* values indicating a higher rate of migration and incorporation into tumor neovascularization of previously cryopreserved CB AC133+ EPCs to glioma sites, compared to non-cryopreserved cells. Conclusion: Magnetically labeled CB EPCs can be in vitro expanded and cryopreserved for future use as MRI probes for monitoring the migration and incorporation to the sites of neovascularization